Immunogenic compositions against tuberculosis

ABSTRACT

Methods of preparing mutants of  Mycobacterium tuberculosis  with one or more disrupted genes are presented, where the disrupted genes include ctpV, rv0990c, rv0971c, and/or rv0348. Compositions containing mutants with attenuated virulence and pathogenesis, which are capable of stimulation of an immune response against  tuberculosis , are described. Compositions and methods relating to immunogenic compositions, which include an attenuated M. tb strain in which the M. tb genome includes a disruption of at least one of the ctpV gene, the rv0990c gene, the rv0971c gene, and the rv0348 gene, are also provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/800,374, filed May 13, 2010, incorporated herein by reference in itsentirety, which claims the benefit of and priority to U.S. ProvisionalApplication No. 61/216,167, filed May 14, 2009, incorporated herein byreference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under AI066235awarded bythe National Institutes of Health. The government has certainr ights inthe invention.

BACKGROUND

The Mycobacterium tuberculosis (“M. tb”) genome is one of the largestbacterial /genomes known, including more than 4 million base pairs andnearly four thousand predicted protein coding sequences. Approximatelyone-third of the world's population is infected with M. tb, thecausative agent of the disease tuberculosis (“TB”) in humans. Infectionwith M. tb is commonly the result of an uninfected person inhaling M. tbbacilli that have become airborne as a result of some action of aninfected person, e.g., coughing, sneezing, spitting, or talkingClinically, infection with M. tb in humans can be divided into threestages.

In the first stage of infection, which typically lasts from three toeight weeks, M. tb bacilli are taken up by alveolar macrophages in thelungs, where they multiply. In the second stage of the infection, whichtypically lasts from two to five months, M. tb multiplies withininactivated macrophages until they burst, whereupon M. tb circulates viathe bloodstream to all body organs including the brain, bone marrow, andother parts of the lung. In the third stage of the infection, whichtypically lasts from six months to two years, the host commonly developsa cell-mediated immune response to M. tb and may experience pleurisyaccompanied by severe chest pain. In the fourth stage of infection,there is either resolution of the primary complex or persistence of theinfection until reactivation, which may occur many years after initialexposure to M. tb. While only 5-10% of non-immunocompromised personsexposed to TB develop active TB during their lives, it is estimated thateach person with active TB infects about 10-15 others annually.Ultimately, TB causes nearly two million deaths every year and is aleading killer of HIV-infected persons.

A vaccine for tuberculosis, Bacille Calmette Guérin (“BCG”), preparedwith an attenuated strain of the bovine pathogen Mycobacterium bovis, isroutinely used world-wide. However, the vaccine utilizes bacteria thatdo not normally cause disease in humans and provides little to noprotection against tuberculosis in adults. Drugs are also available totreat TB, but bacterial resistance has developed against every availabledrug. Moreover, multi-drug resistant (“MDR”) and extensivelydrug-resistant (“XDR”) strains of TB pose a serious threat to humanhealth.

SUMMARY

The present application relates to M. tb mutants, which may exhibitreduced virulence in test subjects as compared to the counterpartwild-type M. tb. As a result of this reduced virulence, the mutantsdescribed herein may be useful for eliciting an immune response in asubject that has been exposed to the mutant. For example, in someembodiments, a pharmaceutically acceptable immunogenic compositioncomprising the M. tb mutants may be administered to a subject. The M. tbmutants described herein are commonly characterized by disruptions inthe ctpV, rv0990c, rv0971c, and/or rv0348 (also known as “mosR”) genesof M. tb.

For example, in some embodiments, engineered Mycobacterium tuberculosis(“M. tb”) strains are provided in which the M. tb genome includes adisruption of at least one of the ctpV gene, the rv0990c gene, therv0971c gene, and the rv0348 gene. In some embodiments, the disruptionresults in a knock-out of the disrupted gene; in other embodiments, thedisrupted gene exhibits decreased expression of the corresponding geneproduct (i.e., RNA, protein). In further embodiments, the disruptionprohibits the transcription of a full-length, wild-type mRNA and/or theproduction of a functional wild-type protein from the disrupted gene.

Gene disruptions may be generated by methods known in the art. Forexample, in some embodiments, the disruption includes an insertion of aheterologous sequence, such as a gene cassette, into the gene. In otherembodiments, the disruption includes the replacement of at least aportion of the wild-type gene sequence with a heterologous sequence,such as a gene cassette. In some embodiments the heterologous sequenceencodes a selectable marker, such as a hygromycin resistance gene.

In some embodiments, the engineered M. tb strains exhibit attenuatedvirulence. For example, in some embodiments, mice infected with theengineered M. tb strain have an increased average post-infectionlifespan compared to mice infected with the corresponding wild-typestrain. For example, in some embodiments, the post infection life spanof mice infected with an engineered attenuated M. tb strain is at leastabout 125% compared to mice infected with the corresponding wild-type M.tb strain. In other embodiments, the post infection life span of miceinfected with an engineered attenuated M. tb strain is at least about125% to about 200% of that of mice infected with the correspondingwild-type M. tb strain. In other embodiments, the post infection lifespan of mice infected with an engineered attenuated M. tb strain is atleast about 130% to about 190%; at least about 140% to about 180%; is atleast about 150% to about 170%; is at least about 160% to about 165%; isat least about 162%; or at least about 138% of the post infection lifespan of mice infected with the wild-type strain. In some embodiments,the engineered attenuated M. tb strain is ΔctpV, Δrv0348, 40990c, orΔ0971c.

In some embodiments, the engineered M. tb strains exhibit a differentresponse to stress as compared to the wild-type counterparts. Forexample, in some embodiments, the average lifespan of an engineered M.tb strain in 500 μM CuCl₂, is decreased by at least about 10% to about50% as compared to a corresponding wild-type M. tb strain. In otherembodiments, the lifespan of an engineered M. tb strain in 500 μM CuCl₂,is decreased by at least about 15% to about 40%; by at least about 20%to about 30%; or by at least about 25% as compared to a correspondingwild-type M. tb strain. In further embodiments, the engineered M. tbstrains exhibit enhanced expression of hypoxia-related genes under lowoxygen conditions as compared to the corresponding wild-type strains. Insome embodiments, the engineered M. tb strain is Δrv0348.

In some embodiments, the engineered M. tb strains exhibit a differentresponse to stress as compared to the wild-type counterparts. Forexample, in some embodiments, the engineered M. tb strains exhibitenhanced expression of hypoxia-related genes under low oxygen conditionsas compared to the corresponding wild-type strains. In some embodiments,the expression of hypoxia-responsive genes under low-oxygen conditionsis not repressed by an rv0348 protein. In some embodiments, theengineered M. tb strain is Δrv0348.

In further embodiments, the engineered M. tb strains exhibit enhancedexpression of one or more of the following genes: Rv0823c-Rv0824c;Rv1622c; Rv1623c; Rv2031c; Rv2629-Rv2630; Rv3048c; and Rv3139-Rv3140. Inother embodiments, the engineered M. tb strains exhibit decreasedexpression of one or more of the following genes: Rv0167-0177;Rv0684-0685; Rv0700-0710; Rv0718-0723; Rv1613-1614; Rv2391, 2392;Rv2948c; Rv3148-Rv3154; Rv3460c; Rv3824c-Rv3825c; Rv3921c-Rv3924c. Insome embodiments, the engineered M. tb strain is Δrv0348.

The present disclosure also relates to immunogenic compositionsincluding engineered M. tb strains. For example, in some embodiments,immunogenic compositions include an attenuated M. tb strain in which theM. tb genome includes a disruption of at least one of the ctpV gene, therv0990c gene, the rv0971c gene, and the rv0348 gene. Some embodiments ofimmunogenic compositions also include a pharmaceutically acceptablecarrier and/or a pharmaceutically acceptable adjuvant.

Also disclosed herein are methods of eliciting or stimulating an immuneresponse in a subject against tuberculosis (e.g., vaccinating a subjectagainst tuberculosis). In some embodiments, an immunogenic compositionincluding an attenuated M. tb strain in which the M tb genome includes adisruption of at least one of the ctpV gene, the rv0990c gene, therv0971c gene, and the rv0348 gene is administered to the subject. Insome embodiments, the subject is a mammal and the immunogeniccomposition is administered orally, nasally, subcutaneously,intravenously or by inhalation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of an in vivo expressed genomic island (“iVEGI”)of M. tb preferentially expressed in murine host during tuberculosis.

FIG. 2 is a representation of the ctpV coding region from which 2.1 kBare deleted (shown in black) to produce the ΔctpV mutant.

FIG. 3 shows Southern blot confirmation of the ΔctpV mutant.

FIG. 4 shows the results of RT-PCR verifying transcription of ctp Vandthe downstream gene rv0970 in the wild-type strain but onlytranscription of the rv0970 gene in the isogenic mutant ΔctpV.

FIG. 5 shows the fold changes in expression of ctpV in H37Rv andisogenic mutants after exposure to 500 μM copper relative to cultureskept copper-free.

FIG. 6 shows growth curves of wild-type M. tb, its isogenic mutantΔctpV, and the complemented strain ΔctpV:ctpV.

FIG. 7 shows growth curves of wild-type M. tb (H37Rv), its isogenicmutant ΔctpV, and the complemented strain ΔctpV:ctpV in the presence of0 μM and 50 μM and 500 μM CuCl₂.

FIG. 8 shows growth curves of the wild-type M. tb in the presence 0 μMand 50 μM and 500 μM CuCl₂ and growth curves of the M. tb isogenicmutant ΔctpV and the complemented strain ΔctpV:ctpV in the presence of500 μM CuCl₂.

FIG. 9 shows the bacterial load of mouse lungs at various times afterinfection with either wild-type M. tb (H37Rv) or its isogenic mutantΔctpV.

FIG. 10 shows survival of mouse groups (N=10) at various times afterinfection with either wild-type M. tb (H37Rv) or its isogenic mutantΔctpV.

FIG. 11 shows acid-fast staining of sectioned mouse lung tissue of miceinfected with wild-type (“WT”) and ΔctpV at two weeks (top) and fourweeks (bottom) post infection.

FIG. 12 shows histological analysis of lung sections of mice lungs at 4weeks (top) and 38 weeks (bottom) of infection with wild-type (“WT”) andΔctpV.

FIG. 13 shows qRT-PCR and microarray data for fold change of expressionof selected genes in ΔctpV relative to wild-type.

FIG. 14 shows a qRT-PCR survey of the response to copper of allpredicated metal-transporting P-type ATPases within the M. tb (H37Rv)genome.

FIG. 15 shows the organization of the rv0348 operon and the strategy forgene disruption.

FIG. 16 shows (B) Southern blot analysis of Sa/I-digested genomic DNA ofthe H37Rv WT and Δrv0348 mutant; (C) PCR analysis of cDNA synthesizedfrom RNA samples purified from H37Rv (lanes 1, 3, 5) or Δrv0348 mutant(lanes 2, 4, 6); and (D) Western blot analysis for different M. tbstrains using polyclonal antibodies raised in rabbits against MBP-rv0348 protein.

FIG. 17 shows growth curves of four different M. tb strains inMiddlebrook 7H9 broth.

FIG. 18 shows lung CFU/GM in murine lungs following aerosol infectionwith H37Rv, Δrv0348, and Δrv0348::rv0348.

FIG. 19 shows survival curves of three mice groups (N=10) infected withH37Rv, Δrv0348, and Δrv0348::rv0348.

FIG. 20 shows histological analysis of lung sections of mice lungs at 2weeks and 20 weeks after infection with H37Rv, Δrv0348 andΔrv0348::rv0348.

FIG. 21 shows CFU/g tissue in murine lungs at time of death for H37Rvwild-type strain (37 weeks) and for Δrv0348 strain (62 weeks).

FIG. 22 shows a transcriptional profile of rv0348 in M. tb (H37Rv) undervariable stressors.

FIG. 23 shows colony counts of four different M. tb strains subjected todifferent stressors.

FIG. 24 shows fold changes of ten genes utilizing RNA from both mutantΔrv0348 and complemented Δrv0348:: rv0348 strains relative to H37Rv wildtype strain.

FIG. 25 shows a comparative analysis of the transcriptome of M. tbexposed to variable conditions.

FIG. 26 shows A) Western blot analysis of the recombinant strain of M.smegmatis mc²155 expressing rv0348 protein; B) the survival curve of M.smeg::pML21 under aerobic and anaerobic conditions (left scale) and foldchange in mosR transcripts as measured by qRT-PCR (right scale); C)Lac-Z repression for constructs for rv3130c promoter; D) Lac-Zrepression for constructs for rv0347; E) Lac-Z repression for constructsfor rv0700; F) Lac-Z induction for constructs for rv0167 (“*” denotessignificant change in a Student's t-Test (p<0.001)).

FIG. 27 shows results of various EMSA assays.

FIG. 28 shows recombinant colonies of M. smegmatis without (left) orwith (right) promoters for the target genes.

FIG. 29 shows CFU/g tissue in murine lungs at various times for H37Rvwild-type strain, Δrv0990c strain, and Δrv0971c strain.

FIG. 30 shows survival curves of three mice groups infected with H37Rvwild-type strain, Δrv0990c strain, and Δrv0971c strain.

FIG. 31 shows the colonization and survival data of ΔctpV and thecorresponding wild-type H37Rv M. tb strain.

FIG. 32 shows histopathology of mouse tissue at 4 weeks post infectionwith ΔctpV and the corresponding wild-type H37Rv M. tb strain.

FIG. 33 shows histopathology at early, chronic stages of mouse tissue at4 weeks post infection with Δrv0971c, Δ0991c and the correspondingwild-type H37Rv M. tb strain.

FIG. 34 shows mouse lung tissue stained with IFN-γ antibody at 8 weekspost infection with wild-type and M.tb mutant strains. The left panelshows mouse lung infected with wild-type M.tb, the middle panel showsmouse lung infected with ΔctpV mutant, and the right panel shows mouselung infected with the ΔctpV:ctpV mutant.

FIG. 35 is a graph showing fold change in expression of csoR at 500 μMcopper in the ΔctpV mutant and the ΔctpV:ctpV mutant relative to csoRexpression in the corresponding WT M. tb strain.

FIG. 36 shows histological analysis of lung sections of mice lungs at 4weeks post infection with wild-type (“WT”) M. tb and ΔctpV andΔctpV::ctpV.

FIG. 37 shows CFU/g tissue in murine lungs at various times for H37Rvwild-type strain, ΔctpV strain, and ΔctpV::ctpV strain.

FIG. 38 shows survival curves of three mice groups infected with H37Rvwild-type strain, ΔctpV strain, and ΔctpV::ctpV strain.

FIG. 39 shows histological (A) and immunohistochemisty (B) analysis oflung sections of mice lungs at 8 or 38 weeks post infection withwild-type (“WT”) M. tb and ΔctpV and ΔctpV::ctpV.

DETAILED DESCRIPTION

The present application relates to novel M. tb mutants which exhibitreduced virulence in test subjects as compared to the wild-type M. tbcounterpart. Due to the reduced virulence, the mutants described hereinare useful for eliciting an immune response in a subject that has beenexposed to the mutant. For example, in some embodiments, the mutants areprovided as a pharmaceutically acceptable immunogenic compound, such asa vaccine.

The novel M. tb mutants described herein are characterized bydisruptions in the ctpV, rv0990c, rv0971c, and/or rv0348 (also known as“mosR”) genes of M. tb.

The present invention is described herein using several definitions, asset forth below and throughout the specification.

As used herein, the term “subject” refers to an animal, preferably amammal, more preferably a human. The term “subject” and “patient” may beused interchangeably.

The term “pharmaceutically acceptable carrier” refers to any carrierthat has substantially no long term or permanent detrimental effect whenadministered to an individual. Pharmaceutically acceptable carriersinclude diluents, fillers, salts, dispersion media, coatings,emulsifying agents, wetting agents, sweetening or flavoring agents,tonicity adjusters, absorption delaying agents, preservatives,antibacterial and antifungal agents, buffers, anti-oxidants,stabilizers, solubilizers, bulking agents, cryoprotectant agents,aggregation inhibiting agents, or formulation auxiliary of any type.Suitable carriers are described in Remington's Pharmaceutical Sciences(Remington's Pharmaceutical Sciences, 2000, 20th Ed., Lippincott,Williams & Wilkins), incorporated herein by reference. Preferredexamples of such carriers or diluents include, but are not limited to,water, sodium chloride, mannitol, trehalose dihydrate, polysorbate 80,various pharmaceutically acceptable buffers for adjusting pH (e.g.phosphate buffers, citrate buffers, acetate buffers, and boratebuffers).

The term “immunogenic composition” is used herein to refer to acomposition that will elicit an immune response in a mammal that hasbeen exposed to the composition. In some embodiments, an immunogeniccomposition includes at least one of four M. tb mutants ΔctpV, Δrv0990c,Δrv0971c, and/or Δrv0348. In some embodiments, the immunogeniccomposition includes at least two, at least three or at least four ofthe mutants ΔctpV, Δrv0990c, Δrv0971c, and/or Δrv0348.

In some embodiments, the immunogenic compositions described herein maybe formulated for administration (i.e., formulated for “exposure” to themammal) in a number of forms. For example, in some embodiments, theimmunogenic compositions are prepared for oral, pulmonary, intravenous,intramuscular, subcutaneous, parenteral, nasal, or topicaladministration. Compositions may also be formulated for specific dosageforms. For example, in some embodiments, the immunogenic composition maybe formulated as a liquid, gel, aerosol, ointment, cream, lyophilizedformulation, powder, cake, tablet, or capsule. In other embodiments, theimmunogenic composition is formulated as a controlled releaseformulation, delayed release formulation, extended release formulation,pulsatile release formulation, and mixed immediate release formulation.In some embodiments, the immunogenic composition is provided as aliquid. In other embodiments, the immunogenic composition is provided inlyophilized form.

The terms “mutation” and “disruption” are used interchangeably herein torefer to a detectable and heritable change in the genetic material.Mutations may include insertions, deletions, substitutions (e.g.,transitions, transversion), transpositions, inversions and combinationsthereof. Mutations may involve only a single nucleotide (e.g., a pointmutation or a single nucleotide polymorphism) or multiple nucleotides.In some embodiments, mutations are silent, that is, no phenotypic effectof the mutation is detected. In other embodiments, the mutation causes aphenotypic change, for example, the expression level of the encodedproduct is altered, or the encoded product itself is altered. In someembodiments, a mutation may result in a disrupted gene with decreasedlevels of expression of a gene product (e.g., protein or RNA) ascompared to the wild-type strain (e.g., M. tb). In other embodiments, amutation may result in an expressed protein with activity that is loweras compared to the activity of the expressed protein from the wild-typestrain (e.g., M. tb).

The term “knockout mutant” is used herein to refer to an organism inwhich a null mutation has been introduced in a gene. In a knockoutmutant, the product encoded by the wild-type gene is not expressed,expressed at levels so low as to have no effect, or is non-functional.In some embodiment, the knockout mutant is caused by a mutation in theknocked out gene. In some embodiments, the knockout mutation isintroduced by inserting heterologous sequences into the gene ofinterest. In other embodiments, the knockout mutation is introduced byreplacing a portion of the wild-type gene or allele, or a majority ofthe wild-type gene or allele, with a heterologous sequence, or anengineered (e.g., manually altered, disrupted, or changed),non-functional, copy of the wild-type sequence.

A “knocked out gene” refers to a gene including a null mutation (e.g.,the wild-type product encoded by the gene is not expressed, expressed atlevels so low as to have no effect, or is non-functional). In someembodiments, the knocked out gene includes heterologous sequences orgenetically engineered non-functional sequences of the gene itself,which renders the gene non-functional. In other embodiments, the knockedout gene is lacking a portion of the wild-type gene. For example, insome emboiments, at least about 10%, at least about 20%, at least about30%, at least about 40% or at least about 60% of the wild-type genesequence is deleted. In other embodiments, the knocked out gene islacking at least about 70%, at least about 75%, at least about 80%, atleast about 90%, at least about 95% or at least about 100% of thewild-type gene sequence. In other embodiments, the knocked out gene mayinclude up to 100% of the wild-type gene sequence (e.g., some portion ofthe wild-type gene sequence may be deleted) but also include one or moreheterologous and/or non-functional nucleic acid sequences insertedtherein.

Generally, a heterologous sequence may be any sequence which does notaffect the expression of other genes in the organism (e.g., does notencode a regulatory protein). Additionally, in some embodiments, aheterologous sequence includes a marker sequence (e.g., a gene that theorganism does not have, and that confers resistance to a drug or otherharmful agent, or that produces a visible change such as color orfluorescence). For example, in some embodiments, the heterologoussequence is the hygromycin resistance gene, as described in Bardarov, etal., 2002, Microbiol.,148:3007-3017, herein incorporated by reference inits entirety. By way of example, but not by way of limitation, othersuitable heterologous sequences include selectable markers such as akanamycin resistance marker or other antibiotic resistance marker,β-galactosidase, or various other detectable markers known to those ofskill in the art.

In a knockout mutant, the heterologous sequence may be expressed (e.g.,may be transcribed and/or translated) or it may be silent (e.g., nottranscribed and/or translated). For example, in the case of M. tbknockout mutants ΔctpV, Δrv0990c, Δrv0971c, and Δrv0348, theheterologous sequence includes the hygromycin resistance gene. Thehygromycin resistance gene is expressed in the knockout mutants, andmutant knockouts are selected, inter alia, by virtue of their ability togrow in the presence of hygromycin.

Mutants, such as knockout mutants may be constructed using methods wellknown in the art, although methods involving homologous recombinationare frequently used. In some embodiments, such methods includetechniques such as electroporation or transduction. In otherembodiments, transposons may be used to disrupt the gene of interestsand insert heterologous sequence. By way of example, but not by way oflimitation, other methods of constructing mutants in M. tb include, forexample, the use of a suicide vector and chemical mutagenesis.

A knockout mutant may include a single knocked out gene or multipleknocked out genes. For example, in some embodiments, an M. tb knockoutmutant includes a knockout of one or more of the following genes: ctpV,rv0990c, rv0971c, and rv0348.

The term “M. tb ΔctpV ,” “ΔctpV mutant,” “ΔctpV knockout,” or “Δctpr isused herein to refer to an M. tb knockout, in which the ctpV gene is notexpressed, expressed at levels so low as to have no effect or theexpressed protein is non-functional (e.g., is a null-mutation). In someembodiments, the ΔctpV mutant includes a heterologous sequence in placeof all or a majority of the ctpV gene sequence. In some embodiments, theheterologous sequence includes the hygromycin resistance gene.

The term “M. tb Δrv0990c,” “Δrv0990c mutant,” “Δrv0990c knockout” or“Δrv0990c” is used herein to refer to an M. tb knockout, in which therv0990c gene is not expressed, expressed at levels so low as to have noeffect or the expressed protein is non-functional (e.g., is anull-mutation). In some embodiments, the Δrv0990c mutant includes aheterologous sequence in place of all or a majority of the rv0990c genesequence. In some embodiments, the heterologous sequence includes thehygromycin resistance gene.

The term “M. tb Δrv0971c,” “Δrv0971c mutant,” “Δrv0971c knockout,” or“Δrv0971c” is used herein to refer to an M. tb knockout, in which therv0971c gene is not expressed, expressed at levels so low as to have noeffect or the expressed protein is non-functional (e.g., is anull-mutation). In some embodiments, the Δrv0971c mutant includes aheterologous sequence in place of all or a majority of the rv0971c genesequence. In some embodiments, the heterologous sequence includes thehygromycin resistance gene.

The term “M. tb Δrv0348,”“Δrv0348c mutant,” “Δrv0348 knockout,”“Δrv0384,” “M tb ΔmosR” “ΔmosR mutant,” “ΔmosR knockout,” or “ΔmosR” isused herein to refer to an M. tb knockout, in which the rv0348 gene isnot expressed, expressed at levels so low as to have no effect or theexpressed protein is non-functional (e.g., is a null-mutation). In someembodiments, the Δrv0348 mutant includes a heterologous sequence inplace of all or a majority of the rv0348 gene sequence. In someembodiments, the heterologous sequence includes the hygromycinresistance gene. As used herein, the term rv0348 and mosR are usedinterchangeably.

The term “vaccine” is used herein to refer to a composition that isadministered to a subject to produce or increase immunity to aparticular disease. In some embodiments, vaccines include apharmaceutically acceptable adjuvant and/or a pharmaceuticallyacceptable carrier.

The term “live attenuated vaccine” is used herein to refer to a vaccineprepared from live bacteria or viruses, which have been weakened so theyproduce immunity when exposed to a subject, but do not cause disease, orcause a less severe form, duration, onset or later onset of the disease.

In some embodiments, a live attenuated vaccine includes at least one ofthe four M. tb knockout mutants ΔctpV, Δrv0990c, Δrv0971c, and Δrv0348.In other live attenuated vaccine embodiments, at least two, at leastthree or at least four of the M. tb knockout mutants are provided. Instill other embodiments, a live attenuated vaccine includes an M. tbknockout that includes multiple “knocked out” genes. For example, insome embodiments, the live attenuated vaccine includes M. tb with aknockout of one or more of the ctpV, rv0990c, rv0971c, and rv0348 genes.

In other embodiments, the “live attenuated vaccine” is a pharmaceuticalcomposition that includes a pharmaceutically acceptable adjuvant and/ora pharmaceutically acceptable carrier.

The term “gene cassette” is used herein to refer to a DNA sequenceencoding and capable of expressing one or more genes of interest (e.g.,a selectable marker) that can be inserted between one or more selectedrestriction sites of a DNA sequence. In some embodiments, insertion of agene cassette results in a disrupted gene. In some embodiments,disruption of the gene involves replacement of at least a portion of thegene with a gene cassette, which includes a nucleotide sequence encodinga selectable marker. In some embodiments, a gene cassette may be anantibiotic resistance gene cassette. In some embodiments, the antibioticresistance gene cassette may be a hygromycin resistance cassette. By wayof example, but not by way of limitation, Bardarov, et al., 2002,Microbiol.,148:3007-3017 describes one embodiment of a hygromycinresistance gene cassette.

The term “engineered” is used herein to refer to an organism that hasbeen deliberately genetically altered, modified, or changed, e.g. bydisruption of the genome. For example, an “engineered M. tb strain”refers to an M. tb strain that has been deliberately geneticallyaltered, modified, or changed.

The term “corresponding wild-type strain” or “parent wild-type strain”is used herein to refer to the wild-type M. tb strain from which theengineered M. tb strain was derived. As used herein, a wild-type M. tbstrain is a strain that has not been engineered to knock out one or moreof the ctpV, rv0990c, rv0971c, or rv0348 genes. The engineered M. tbstrain may have been modified to knock out more than one of the ctpV,rv0990c, rv0971c, or rv0348 genes.

The term “pathogen” or “infectious agent” is used herein to refer to aspecific causative agent of disease or illness in a host, such as, forexample, a bacterium or virus.

The term “strain” is used herein to refer to a genetic variant of aorganism, such as bacteria or virus. Thus, a wild-type M. tb strain isgenetically different from a mutant M. tb strain.

The term “attenuated strain” is used herein to refer to a strain withweakened or reduced virulence in comparison to the correspondingwild-type strain.

The term “post-infection lifespan” (“PILS”) is used herein to refer tothe length of time an organism survives (i.e., lives) after infectionwith an infectious agent (e.g., an M. tb strain). As used herein, thePILS of an organism infected with a “standard” or “reference” infectiousagent (e.g., a wild-type M. tb strain) is 100% when compared to the PILSof an organism infected with a “test” infectious agent (e.g., anengineered mutant strain of the “standard” or “reference” infectiousagent). A PILS of greater than 100% indicates the organism infected withthe test infectious agent lives longer than the organism infected withthe reference infectious agent. A PILS of less than 100% indicates thatthe organism infected with the test infectious agent lives less long(i.e., dies sooner) than the organism infected with the referenceinfectious agent. In some embodiments, the “infected organism” is amouse, and the infectious agent is an M. tb strain. In some embodiments,the “reference” M. tb strain is a wild-type M. tb strain and the “test”infectious agent is an engineered mutant of the wild-type M. tb strain.

The term “average post-infection lifespan” refers to the average time agroup of organisms survives post-infection.

In some embodiments, the post-infection lifespan of organisms infectedwith different infectious agents (e.g., different strains of M. tb) arecompared. For example, in some embodiments, the PILS of an organism(e.g., a mouse) infected with an engineered M. tb strain (i.e., “test”strain) is compared to the PILS of an organism (e.g., a mouse) infectedwith the corresponding wild-type strain of M. tb (i.e., the “reference”strain). In some embodiments, the average post-infection lifespan oforganisms infected with different infectious agents (e.g., differentstrains of M. tb) are compared. For example, in some embodiments, theaverage PILS of mice infected with an engineered M. tb strain iscompared to the average PILS of mice infected with the correspondingwild-type strain of M. tb. In some embodiments, the medianpost-infection lifespan of organisms infected with different infectiousagents (e.g., different strains of M. tb) are compared. For example, insome embodiments, the median PILS of mice infected with an engineered M.tb strain is compared to the median PILS of mice infected with thecorresponding wild-type strain of M. tb.

By way of example, but not by way of limitation, the median PILS of miceinfected with a wild-type M. tb reference strain is 29 weeks. Incomparison, mice infected with a mutant M. tb strain, (e.g., ΔctpV) havea median PILS of 47 weeks. In this example, the PILS of mice infectedwith the mutant M. tb is at least 62% greater than the PILS of miceinfected with the wild-type M. tb reference strain. Thus, the PILS ofthe mice infected with the mutant M. tb is 162% of the PILS of miceinfected with the wild type M. tb. As another non-limiting example, miceinfected with mutant M. tb strain Δrv0348 live for at least 40 weeks,while mice infected with the corresponding wild-type M. tb strain have amedian survival time of 29 weeks. Accordingly, the PILS of mice infectedwith the mutant M. tb strain is at least 138% of mice infected with thewild-type strain.

The term “virulence” is used herein to refer to the relative ability ofa pathogen to cause disease.

The term “attenuated virulence” or “reduced virulence” is used herein torefer to a reduced relative ability of a pathogen to cause disease. Forexample, attenuated virulence or reduced virulence can describe bacteriaor viruses that have been weakened so they produce immunity when exposedto a subject, but do not cause disease, or cause a less severe form,duration, onset or later onset of the disease.

The term “pathogenesis” is used herein to refer to the series of eventsleading up to a disease and the step-by-step development of the diseasedue to structural and/or functional changes to a cell, tissue, or organcaused by a pathogenic agent (e.g., bacterium, virus, chemical compoundetc.).

The term “attenuated pathogenesis” is used herein to refer to areduction in the number or severity of events leading up to a disease ora slowing of the development of a disease.

The term “adjuvant” is used herein to refer to a substance that enhancesthe pharmacological effect of a drug or increases the immune response toan antigen. By way of example, but not by way of limitation, adjuvantsinclude mineral salts, e.g., aluminium hydroxide and aluminum or calciumphosphate gels; oil emulsions and surfactant based formulations, e.g.,MF59 (microfluidised detergent stabilized oil-in-water emulsion), QS21(purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion);particulate adjuvants, e.g., virosomes (unilamellar liposomal vehiclesincorporating influenza haemagglutinin), ASO4 ([SBAS4] Al salt withMPL), ISCOMS (structured complex of saponins and lipids), polylactideco-glycolide (PLG); microbial derivatives (natural and synthetic), e.g.,monophosphoryl lipid A (MPL), Detox (MPL+M Phlei cell wall skeleton),AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidalimmunostimulators able to self organize into liposomes), OM-174 (lipid Aderivative), CpG motifs (synthetic oligonucleotides containingimmunostimulatory CpG motifs), modified LT and CT (genetically modifiedbacterial toxins to provide non-toxic adjuvant effects), endogenoushuman immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can beadministered either as protein or plasmid encoded), Immudaptin (C3dtandem array); inert vehicles, such as gold particles.

As described above, the present application provides novel knockoutmutants of Mycobacterium tuberculosis (“M. tb”) useful in eliciting animmune response in a mammal against M. tb. The following examples arepresented to illustrate 1) methods of producing knockout mutants, 2)methods of testing virulence of a knockout, and 3) methods of elicitingan immune response with the mutants. The examples are provided to assistone of ordinary skill in making and using the same. The examples are notintended in any way to otherwise limit the scope of the invention. Thesection is divided into eight main Examples: Example I providesinformation regarding the ΔctpV knockout mutant; Example II providesinformation regarding the Δrv0384 knockout mutant; Examples III providesinformation regarding a Δrv0990c knockout mutant; Example IV providesinformation regarding a Δrv0971c knockout mutant; Example V providesinformation regarding M. tb. infected mice; Example VI providesinformation regarding mice infected with a Δrv0990c mutant; Example VIIprovides information regarding mice infected with a Δrv0971c mutant; andExample VIII provides information regarding the use of M. tb mutants togenerate an immune response and as vaccines.

EXAMPLE I Δctp V Knockout Mutant

A. Overview

Many enzymes require a metal cofactor for activity. The metals thatserve as cofactors in enzymes required for life are consideredbiologically active metals. These metals, including iron, copper, zinc,and magnesium, serve as required micronutrients for many diversecellular organisms, from humans to bacteria. For bacteria that colonizethe human body, this can serve as a form of environmental stress, asmicrobes and host cells struggle for the possession of the samemicronutrients.

The most commonly used metal cofactor, iron, has frequently been studiedin the context of this struggle. Required by both host cells andbacteria for a number of enzymatic activities, including respiration anddetoxification, iron is kept bound by host proteins such as transferrinand lactoferrin. Successful human pathogens have developed compoundstermed “siderophores” to compete with host proteins for bound iron.These microbes also use iron-specific uptake mechanisms and regulatorswhich contribute to iron scavenging and survival within a host.

Another biologically active metal, copper, is also required by both hostand bacterial enzymes, including oxidases and superoxide dismutase. Thepotential role of copper in host/microbe interactions has not yet beenelucidated. Interestingly, studies of copper homeostasis mechanisms inpathogenic organisms have shown that copper export, as opposed toacquisition, seems to be most important for virulence. For example,copper export is required for full virulence of the human pathogensPsuedomonas aeruginosa and Listeria monocytogenes as well as the plantpathogen Pseudomonas fluorescens. Conversely, no copper importers oruptake mechanisms have been identified as required for virulence in anypathogen.

Studies of biometals indicate that although pathogenic bacteria mustobtain sufficient amounts of micronutrients, they are also sensitive tometal toxicity. Therefore, an equal balance of metal import and export(homeostasis) must be obtained at levels appropriate for each metal.Presumably, the ability to sense metals in the environment and regulatethe expression of homeostasis mechanisms is key to maintainingintracellular metals at appropriate concentrations.

The first copper-binding transcriptional regulator in Mycobacteriumtuberculosis (“M. tb”) was identified, and it has been demonstrated thatthis important human pathogen has the ability to respond to copper inits intracellular environment. As shown in FIG. 1, this copper-bindingregulator, CsoR, is encoded within a region of the M. tb genomepreviously associated with in vivo survival, an in vivo expressedgenomic island (“iVEGI”) of M. tb preferentially expressed in murinehost during tuberculosis. CsoR has been shown to upregulate theexpression of its own operon, the cso operon, in response to increasingamounts of copper. The cso operon includes the ctpV gene, which encodesa putative metal transporter previously associated with copper responsein M. tb.

Previously, the expression of the ctpV gene was determined to be inducedby copper ions via the copper-binding transcriptional regulator CsoR.ctpV was identified as a member of the M. tb whole-genometranscriptional response to copper at both growth-permissive and toxicphysiological levels, with highest induction occurring at toxic copperlevels. Sequence analysis showed that ctpV has ˜70% protein-levelsimilarity to previously characterized copper transporters involved incopper export and import in Escherichia coli and Enterococcus hirae,respectively. Thus, due to its particularly high induction duringexposure to toxic levels of copper, ctpV may encode a copper exporterrequired for detoxification in the presence of elevated copper.

In the following example, the role of ctpV in mycobacterial copperresponse and its relevance to the development of tuberculosis is shown.CtpV is a copper exporter required for copper homeostasis in M. tb. TheCtpV copper exporter is also required for the full virulence of thebacteria in a mouse model of infection.

B. Construction of the ctpV Knockout Mutant

A knockout mutant of ctpV, ΔctpV, was created in the virulent M. tbstrain H37Rv by replacing the coding region of ctpV with a hygromycinresistance cassette using homologous recombination. Referring to FIG. 2,the ΔctpV mutant was created with homologous recombination via pML21, aderivative of pPR27, which resulted in the deletion of 2.1 kB of thectpV coding region (represented in black) and the insertion of a 3.5 kBregion encoding a hygromycin resistance cassette. The ctpV gene is 2313base pairs long. About 2.1 kB of the coding sequence of ctpV (base 102to 2239) were deleted from the genome of M. tb and replaced by thehygromycin cassette.

Briefly, to construct ΔctpV, 800 basepair fragments of both the upstreamand downstream portion of the gene were amplified by PCR (primersAMT567, AMT568, AMT371, and AMT372are listed in Table 3). The amplifiedfragments were cloned into the pGEM-T Easy vector (Promega, Madison,Wis.). The fragments were digested with the flanking restriction enzymesites (AflII/XbaI and HindIII/SpeI for upstream and downstream portions,respectively) and ligated into pYUB854. After digestion by NotI and SpeI(Promega), the linearized vector was ligated into pML19, a derivative ofpPR27 where a kanamycin resistance cassette has been inserted into thePstI site. The resulting vector was named “pML20”.

This vector was electroporated into electrocompetent M. tb using a GenePulser II machine (BioRad, Herculese, Calif.), and cells were platedonto Middlebrook 7H10 supplemented with 10% albumin-dextrose-catalase(ADC) and 50 ug/mL hygromycin (Invitrogen, Carlsbad, Calif.). After onemonth of growth at 32° C., transformants were grown for two weeks withshaking at 32° C. in Middlebrook 7H9 supplemented with 10% ADC and 50ug/mL hygromycin. These cultures were plated onto Middlebrook 7H10supplemented with 10% ADC, 2% sucrose, and 50 ug/mL hygromycin andincubated at 39° C. for three weeks. The genomic incorporation of theplasmid was confirmed via the inability of colonies to grow onMiddlebrook 7H10 supplemented with 25 ug/mL kanamycin.

The transformant used for experiments, ΔctpV, was confirmed via negativePCR for the ctpV coding region and positive PCR for the hygromycinresistance cassette with primers listed in Table 3, AMT885, AMT886,AMT887, and AMT926.

Additionally, Southern blot analysis was performed on ΔctpV and wildtype genomic DNA (5 μg) digested with BamHI (Promega), using probes forthe remaining coding region of ctpV or the hygromycin resistancecassette. Referring to FIG. 3, the mutant was confirmed with Southernblots using a membrane constructed from BamHI-digested genomic DNA forWT or ΔctpV. Incubation with a P-32 labeled probe for the remaining ctpVregion (FIG. 3, left) revealed the increased size of the band in ΔctpVresulting from the loss of two BamHI restriction enzyme sites within thectpV coding region, as shown in FIG. 2, when it was replaced with HygR,which contains no BamHI sites. Additionally, a probe for the hygromycinresistance cassette (FIG. 3, right) hybridized only to the mutant gDNA.

Because ctpV is the third gene in the 4-gene cso operon, ΔctpV wastested for possible polar effects on the downstream gene of unknownfunction, rv0970. Using reverse-transcriptase PCR, the transcription ofrv0970 in the mutant strain was confirmed. Referring to FIG. 4, thepolarity of the ctpV knockout mutant was addressed using RT-PCR to checkfor transcription of its downstream gene. In the wild-type strain(left), positive bands show that ctpV and the downstream gene rv0970 areboth encoded in the genome and transcribed (able to be amplified fromcDNA), with negative amplification from RNA shown as a negative control.In the isogenic mutant ΔctpV (right), the ctpV coding region is notpresent in the genome nor is it transcribed, but the downstream generv0970 is unaffected.

C. Construction and Evaluation of a ctpV Complement

A complemented strain was created by cloning the ctpV coding region intoan integrative vector (pMV361) containing the constitutive hsp60promoter and transforming into the ΔctpV mutant strain. Integration ofctpV into the ΔctpV genome to create the complemented strain ΔctpV:ctpVwas confirmed with PCR, and restored gene expression was confirmed withqRT-PCR.

Briefly, for complementation of ΔctpV, the ctpV coding region wasamplified and cloned into the pGEM-T easy vector for sequencing. ThepGEM vector was then digested with EcoRI and HindIII (Promega), and thefragment was ligated into pMV361. The vector was sequenced, and thenelectroporated into electrocompetent ΔctpV cells and plated on 7H10supplemented with 10% ADC with 50 ug/mL hygromycin and 25 ug/mLkanamycin. The complemented strain was confirmed using a forward primerwithin the pMV361 vector (hsp60) and a reverse primer within the ctpVcoding region.

D. Evaluation of ΔctpV, Complement ΔctpV:ctpV and Wild-Type M. tb StrainH37Rv

Referring to FIG. 5, expression of ctpV was measured using qRT-PCR, withcDNA created from 30 mL cultures exposed to 500 μM Cu. Data is expressedas fold-change relative to cultures kept copper-free, a condition inwhich expression from the cso operon is minimal. qRT-PCR did not detectexpression of ctpV in the knockout mutant (middle), but expression wasrestored in the complemented strain (right). The higher level ofinduction in the complemented strain relative to WT H37Rv (left) isindicative of the increased strength of the hsp60 promoter used forcomplementation relative to the native cso promoter.

A growth curve in 7H9 media revealed that ΔctpV and its complementΔctpV:ctpV have no generalized growth defects relative to WT. Referringto FIG. 6, growth curves of WT, its isogenic mutant ΔctpV, and thecomplemented strain ΔctpV:ctpV were performed in 7H9+ADC. 7H9 media wasprepared using Remel brand 7H9 Middlebrook powder, prepared as describedby manufacturer. 10% ADC supplementation consisted of adding 100 mLcontaining 2 grams glucose, 5 grams BSA fraction V, and 0.85 grams NaClto 900 mL 7H9 media. Cultures were seeded from stock to OD 0.10 andallowed to grow for 14 days at 37° with shaking, with CFUs taken at 0,4, 8, and 14 days via plating on 7H10+ADC. The data show an identicalgrowth rate among the three strains. Two biological replicates wereperformed.

CsoR is induced proportionally to intracellular copper concentration.The ΔctpV mutant shows increased expression of csoR at 500 μM copper ascompared to wild-type, while the ΔctpV:ctpV mutant shows decreasedexpression of csoR at 500 μM copper. (See FIG. 35).

The ΔctpV mutant and the complemented strain ΔctpV:ctpV were then usedto experimentally characterize the role of ctpV in copper response.

E. ctp V Expression is Required for Optimal Survival in High Copper

To test the role of ctpV in copper transport, copper sensitivity betweenΔctpV and wild-type H37Rv M. tb (“WT”) were compared. The knockout of acopper exporter would be expected to result in increased sensitivity tocopper-based toxicity, and this phenotype has been observed in knockoutsof previously characterized copper exporters in other organisms. To testthe copper sensitivity phenotype of ΔctpV, growth curves of WT and ΔctpVin liquid broth cultures supplemented with defined amounts of copperwere performed using a range of copper concentrations previouslydetermined to be physiologically relevant.

Growth curves were performed in 30 mL Cu-free Sauton's minimalmedia+0.05% Tween, prepared using water treated with Chelex(Sigma-Aldrich, St Louis, Mo.), with defined amounts of CuCl₂ added.Glassware was acid-washed (1N nitric acid) to maintain metal-freeconditions. Cultures were seeded to OD 0.1 with bacterial stock washed2× in Sauton's, and allowed to grow for 14 days at 37° C. with shakingColony forming units (“CFUs”) were determined at 0, 4, 8, and 14 dayspost-exposure by plating on Middlebrook 7H10+10% ADC, with 50 ug/mLhygromycin added in the case of the mutant and complemented strains.

Growth at 0 μM and 50 μM CuCl₂ were identical among the three strains,as shown in FIG. 7, thus only data for WT at 0 μM and 50 μM CuCl₂ areshown in FIG. 8. Referring to FIG. 8, at 500 μM copper, ΔctpV displayedan increased copper sensitivity relative to WT, while the complementedstrain showed a decreased copper sensitivity. Comparisons of CFUs ofΔctpV and WT strains revealed that at toxic levels of copper (500 μMCuCl₂), the ΔctpV strain, lacking ctpV, survived for 8 days, while theWT strain survived for 14 days.

F. ctp V Expression is Required for Virulence of M. tb

The ctpV gene is part of a 29-gene genomic island called the in-vivoexpressed genomic island (“iVEGI”) previously shown to be preferentiallyinduced in mice relative to in vitro culture. The ctpV gene may play arole in the survival of M. tb within a host, as experiments have shownthat ctpV is a copper exporter, and data indicate that copperhomeostasis in bacteria may play a role in pathogenesis, though this hadnot previously been tested in M. tb.

BALB/c mice were infected with either ΔctpV or wild-type M. tb using alow-dose aerosolization protocol. Bacterial survival and mouse lungpathology were measured at short-term as well as long-term time pointsvia the homogenization and plating of infected lung tissue as well asorgan histology. Additionally, mice infected with the two strains weremonitored over the long-term course of the infection and the survival ofthe infected mice was recorded.

Briefly, BALB/c mice (Harlan, Indianapolis, Ind.) were infected in aGlas-Col chamber (Glas-Col, LLC, Terra Haute, Ind.) loaded with 10 mL ofeither ΔctpV or wildt-type at OD 0.30. Infectious dose of approximately300 CFU/animal was confirmed via a 1-day time point. CFUs weredetermined by homogenizing lung tissue in PBS buffer and plating onMiddlebrook 7H10+10% ADC, followed by incubation at 37° C. for onemonth. Final CFUs were normalized to the weight of the lung tissue used.Sections of lung, liver, and spleen tissue were taken and incubated informalin prior to sectioning and staining with H&E and AFS.Histopathology slides were examined and scored by a pathologist notassociated with the study.

As shown in FIG. 9, a decrease in lung CFUs of ΔctpV relative to WT wasobserved at both short-term and long-term time points. Referring to FIG.9, the bacterial load of mouse lungs after infection with either wildtype (H37Rv) or its isogenic mutant ΔctpV was determined viahomogenization of lungs from infected mice (N=3-5 per time point) in PBSand plating on 7H10+ADC (numbers normalized to grams of lung tissuehomogenized) with approximately one-log difference seen between ΔctpVand WT at 2 weeks and 38 weeks. Mice infected with ΔctpV lived longerthan mice infected with WT, with a 18-week increase in time to death, asshown in FIG. 10. Referring to FIG. 10, the survival of mouse groups(N=10) after infection with WT or ΔctpV is shown. The median survivaltime for mice infected with WT was 29 weeks, versus 47 weeks for miceinfected with ΔctpV.

As shown in FIG. 11, acid-fast stains of histology sections confirm thelower bacterial load of mice infected with ΔctpV compared to infectionwith the wild-type strain at two weeks post infection. As shown in FIG.12 and FIG. 36 (4 weeks post infection comparison of mouse lung tissueinfected with wild-type, Δctpv and the ΔctpV::ctpv M. tb complement),histopathology revealed lower levels of tissue destruction along thecourse of infection with ΔctpV relative to WT, even at time points wherebacterial load was not significantly different between ΔctpV and WT. At8 weeks post infection, lung tissue from mice infected with ΔctpVdisplayed granulomatous inflammation, whereas mice infected with H37Rvdisplayed massive granulomatous inflammation with more lymphocyticinfiltration. And by 38 WPI, granuloma formation occupied almost thewhole lungs of mice infected with the wild-type strain, compared to only50% of tissues of mice infected with the ΔctpV mutant. However, whetherwild type or the ΔctpV were used for infection, granulomatous lesionsincluded sheets of lymphocytes and aggregates of activated macrophages.Leisons observed in the complemented strain were very similar to thoseobserved in mice infected with the H37Rv strain.

Immunohistochemstry using IFN-γ antibodies was performed on lung tissuesections from mice infected with WT, ΔctpV orΔctpV::: ctpV M. tbstrains. At eight weeks post infection, mice infected with the ΔctpVmutant showed decreased IFN-γ expression relative to mice infected withthe corresponding wild-type M. tb strain. The IFN-γ antibody appearsbrown in FIG. 34.

Taken together, mouse infection data indicates a role for ctpV in M.tbsurvival in lungs as well as overall virulence and host mortality.

G. Other Genes are Expressed under High Copper Conditions in the ctpVKnockout

In M. tb, ctpV is very highly expressed at toxic concentrations ofcopper (500 μM), and it has been shown that ctpV is required for normalsurvival at this level of copper. Interestingly, the genome of M. tbencodes a number of genes with high sequence similarity to ctpV.Specifically, ctpV is a metal translocation P-type ATPase, and there areten other predicted metal-translocating P-type ATPases in the H37Rvgenome with significant sequence similarity to ctpV as shown in Table 1.With the exception of ctpV, potential cation prediction is based solelyon sequence data. Percent similarity was determined at the protein levelusing MATCHER. Sequences were obtained from Tuberculist.

TABLE 1 Predicted Metal-Translocating P-type ATPases in the H37Rv Genomewith Significant Sequence Similarity to ctpV Gene name Product namePotential Cation % similarity to CtpV rv0092 CtpA Copper 65.3 rv0103cCtpB Copper 61.4 rv3270 CtpC Unknown 54.7 rv1469 CtpD Cadmium 50.4rv0908 CtpE Unknown 44.5 rv1997 CtpF Unknown 44.7 rv1992c CtpG Unknown55.5 rv0425c CtpH Unknown 44.1 rv0107c CtpI Magnesium 44 rv3743c CtpJCadmium 49.8 rv0969 CtpV Unknown —

The presence of redundant proteins is indicated by the delayed coppersensitivity phenotype as shown in FIG. 10. The eight-day delay in aphenotypic difference between WT and ΔctpV implies that, initially,other mechanisms may be able to compensate for the lack of ctpV,although any such functional complementation is only partial after eightdays.

Cultures of Δctp V were exposed to 500 μM CuCl₂ for 24 hours andtranscript levels of the cells were compared to those of wildtypecultures that had been exposed to the same conditions, as publishedpreviously (see e.g., Ward, et al., J Bacteriol 2008 April; 190(8):2939-46).

Briefly, cultures of ΔctpV were inoculated to OD 0.1 in Sauton's mediaand allowed to grow shaking at 37° C. to OD 0.6. The cultures were thensupplemented with 500 μM CuCl₂ and incubated for three more hours priorto spinning down the cultures and freezing immediately at −80° C.

Briefly, RNA was extracted using a Trizol-based method (Invitrogen,Carlsbad, Calif.), and treated with DNAse I (Ambion, Austin, Tex.) toremove contaminating DNA. cDNA was synthesized from 1 μg total RNA usingan Invitrogen SuperScript ds-cDNA synthesis kit in the presence of 250ng genome-directed primers. cDNA clean up, Cy3 labeling, hybridizations,and washing steps were performed using the NimbleGen gene expressionanalysis protocol (NimbleGen Systems, Inc., Madison, Wis.). Microarraychips were purchased from NimbleGen Systems, Inc., and they containednineteen 60-mer probes for each of the 3,989 open reading framesidentified in the genome of M. tb H37Rv, with five replicates of thegenome printed on each slide (total of 95 probes/gene). Slides werescanned using an Axon GenePix 4000B scanner (Molecular DevicesCorporation, Sunnyvale, Calif.), and fluorescence intensity levelsnormalized to 1000. Significantly changed genes between WT and ΔctpVwere determined using the EBArrays package in R (R is an open sourceplatform used by Bioconductor, an open source and open developmentsoftware project). A cutoff value of 0.50 for the probability ofdifferential expression, determined using an LNN model, was used todetermine statistically differentially expressed genes.

Ninety-eight genes with significantly different expression levelsbetween ΔctpV and WT after exposure to 500 μM CuCl₂ were identified andare listed in Table 2.

TABLE 2 Genes with Significantly Different Expression Levels betweenΔctpV and WT after Exposure to 500 μM CuCl₂ rv name ΔctpV500/wt500 Genename Description Gene Regulation rv1221 1.86 sigE ECF subfamily sigmasubunit rv1379 1.57 pyrR regulatory protein - pyrimidine biosynthesisrv1398c 2.14 conserved hypothetical protein rv1909c 4.46 furA ferricuptake regulatory protein rv1994c 2.98 transcriptional regulator (MerRfamily) rv3260c 1.81 whiB2 WhiB transcriptional activator homologueTransporters rv0969 −2.45 ctpV cation transport ATPase rv2398c −2.14cysW sulphate transport system permease protein Membrane/secretedproteins rv0451c −2.83 mmpS4 conserved small membrane protein rv0970−3.07 hypothetical protein rv1566c 1.54 putative exported p60 proteinhomologue rv1799 1.52 lppT probable lipoprotein rv1980c −2.47 mpt64secreted immunogenic protein Mpb64/Mpt64 rv1987 1.90 probable secretedprotein rv2080 2.75 lppJ lipoprotein rv3763 1.86 lpqH 19 kDKD Enzymesrv0247c −1.72 probable iron-sulphur protein *(succinate dehydrogenase)rv0462 −1.73 probable dihydrolipoamide dehydrogenase rv1182 −2.21 papA3PKS-associated protein, unknown function rv1185c −1.78 fadD21 acyl-CoAsynthase rv1471 1.89 trxB thioredoxin reductase rv1520 1.58glycosyltransferase rv1908c −1.68 katG catalase-peroxidase rv2196 −1.82qcrB cytochrome b component of ubiQ-cytB reductase rv2200c −1.58 ctaCcytochrome c oxidase chain II rv2244 2.00 acpM acyl carrier protein(meromycolate extension)*(polyketide/fatty acid biosynthesis) rv2445c−1.58 ndkA nucleoside diphosphate kinase rv2930 −1.75 fadD26 acyl-CoAsynthase rv3116 −1.55 moeB molybdopterin biosynthesis rv3117 −1.61 cysA3thiosulfate sulfurtransferase rv3146 −1.62 nuoB NADH dehydrogenase chainB rv3359 1.59 probable oxidoreductase rv3377c −1.68 similar to manycyclases involved in steroid biosynthesis rv3824c −1.80 papA1PKS-associated protein, unknown function Mce proteins rv0169 −1.58 mce1cell invasion protein rv0170 −1.87 part of mce1 operon rv0171 −1.59 partof mce1 operon rv0174 −1.57 part of mce1 operon Ribosomal proteinsrv0055 3.29 rpsR 30S ribosomal protein S18 rv0056 1.75 rplI 50Sribosomal protein L9 rv0710 1.52 rpsQ 30S ribosomal protein S17 rv0719−1.65 rplF 50S ribosomal protein L6 rv1298 2.11 rpmE 50S ribosomalprotein L31 rv2882c −1.65 frr ribosome recycling factor Protein faterv2109c −2.13 prcA proteasome [alpha]-type subunit 1 rv2457c −1.77 clpXATP-dependent Clp protease ATP-binding subunit ClpX rv2903c 1.83 lepBsignal peptidase I rv2094c 3.74 tatA tatA subunit tatAB secretion systemrv3875 2.09 esat6 early secretory antigen target Other rv3841 1.75 bfrBbacterioferritin rv0001 −1.61 dnaA chromosomal replication initiatorprotein rv1080c −1.80 greA transcription elongation factor GHypothetical proteins rv0021c 1.83 conserved hypothetical protein rv01401.54 conserved hypothetical protein rv0236A −1.53 rv0500B 1.81 rv05081.55 hypothetical protein rv0664 −1.52 hypothetical protein rv0686 −1.72potential membrane protein rv0730 1.60 conserved hypothetical proteinrv0740 −1.52 conserved hypothetical protein rv0755A 1.90 rv0759c 1.62conserved hypothetical protein rv0991c 1.60 hypothetical protein rv1087A1.51 rv1134 1.61 hypothetical protein rv1334 1.58 conserved hypotheticalprotein rv1501 1.97 conserved hypothetical protein rv1532c 1.51conserved hypothetical protein rv1765A 2.35 rv1783 −2.18 conservedhypothetical protein rv1794 −2.07 conserved hypothetical protein rv18102.26 conserved hypothetical protein rv1982c 1.69 conserved hypotheticalprotein rv2269c 1.57 hypothetical protein rv2401 1.56 hypotheticalprotein rv2623 −1.58 conserved hypothetical protein rv2632c 1.55conserved hypothetical protein rv2706c 1.77 hypothetical protein rv2708c1.51 conserved hypothetical protein rv2804c 1.74 hypothetical proteinrv2970A 1.69 rv3022A −2.33 rv3131 −1.73 conserved hypothetical proteinrv3142c 2.34 hypothetical protein rv3221A 1.99 rv3222c 1.74 conservedhypothetical protein rv3288c −1.90 conserved hypothetical proteinrv3395A 2.24 rv3412 1.87 conserved hypothetical protein rv3492c −1.57conserved hypothetical protein rv3528c 1.90 hypothetical protein rv3614c−1.65 conserved hypothetical protein rv3616c 2.53 conserved hypotheticalprotein rv3633 −1.94 conserved hypothetical protein rv3658c 1.63probable transmembrane protein rv3686c 2.01 conserved hypotheticalprotein rv3822 −1.52 conserved hypothetical protein

To confirm the validity of the microarray data, expression levels ofnine of the genes identified in the microarray dataset were tested withqRT-PCR.

Briefly, qRT-PCR was performed using a SYBR green-based protocol. cDNAwas synthesized from DNAse-treated RNA, obtained as described above,using SuperScript III (Invitrogen) as directed by the manufacturer, inthe presence of 250 ng mycobacterial genome-directed primers. 100 ngcDNA was used as template in a reaction with iTaq SYBR green Supermixwith ROX (Bio-Rad Laboratories, Hercules, Calif.) in the presence ofgene-specific primers (see Table 3) at a concentration of 200 nM. Cycleconditions were 50° C. for 2 min, 95° C. for 3 min, and 40 cycles of 95°C. for 15 seconds and 60° C. for 30 seconds. Reactions were performed intriplicate on an AB7300 machine (Applied Biosystems, Foster City,Calif.) with fluorescence read at the 60° C. step. Threshold cyclevalues were normalized to 16S rRNA expression.

TABLE 3 Gene-Specific Primers Primer Sequence Purpose AMT371ATCACTACTAGTTGAAGACGGTTCGGGGCCAT ctpV flank cloning AMT372ATCACTAAGCTTATAGGCGTGCACGGCGTGCA ctpV flank cloning AMT567ATCACTTCTAGAGGGTTCTCCTCGGTCAGCGTG ctpV flank cloning AMT568ATCACTGGTACCCAGAAACGTCCGCCCCGCTG ctpV flank cloning AMT874GCGGGTGTGGTTGGCCTTGCCGTT Internal primer for mutant screening AMT875GCGGCAACGATCGCCGCACCGATG Internal primer for mutant screening AMT926TGGTGGACCTCGACGACCTGCAGG ctpV mutant screening AMT887ACGAAGCGCGCGAAGGGATGCTGG ctpV mutant screening AMT885GGAACTGGCGCAGTTCCTCTGGGG ctpV mutant screening AMT886TTGACCGCAAAGAAGCGCGCGGCG ctpV mutant screening AMT1335 ACCTCGAACATGGACACctpV Forward AMT1336 ACCGGCAAACAACTGATAC ctpV Reverse AMT1114CAATCCAGGGAAATGTCA esxA Forward AMT1115 AGCTTGGTCAGGGACT esxA ReverseAMT1116 TCGTTGGGCAAGTCAT tatA Forward AMT1117 GCTTCCGCTTTGTTCTtatA Reverse AMT1132 AACCCGGTGGCAAACAAC ctaC Forward AMT1133CGCAGTGGCCCACGAATG ctaC Reverse AMT1168 CCGTCCTGGAAGCAGTGAATGfurA Forward AMT1169 AAACGCACGGCACCGAAA furA Reverse AMT1178GCCGCACAGTTCAACGAAAC rv1471 Forward AMT1179 CGCACCAGGAGGCCCAATrv1471 Reverse AMT1194 AGCACGATGCCGAAGACCTG sigE Forward AMT1195TGCCCGGCTGGTAATTCTG sigE Reverse AMT1196 GTCGAGGAACGAAACCATGCAATbfrB Forward AMT1197 ACCGTGTCTACGCCGGGAAT bfrB Reverse AMT1198CGCGGCGATGAACGACAT qcrB Forward AMT1199 ACGGCGGCAGAATCACCAT qcrB Reverse

The microarray fold-change direction was confirmed by qRT-PCR for allnine genes, as shown in FIG. 13. Referring to FIG. 13, from left toright are: esxA, tatA, ctpV, ctaC, furA, rv1471, sigE, bfrB, and qcrB.qRT-PCR was used to confirm selected genes from the microarray data set,using RNA from the original microarray experiment as the source forcDNA. Direction of induction was reproduced in qRT-PCR data 100% of thetime with data shown as fold change of expression in ΔctpV relative toWT and normalized to 16S expression. Additionally, qRT-PCR of cDNA fromΔctpV:ctpV cells treated with the same 3-hour exposure to 500 μM coppershowed that complementation restored WT-levels of induction for selectedgenes from the microarray data set.

Because the removal of a copper exporter increases intracellular copperconcentration, it was expected that the ΔctpV response to toxic copperlevels relative to the response of WT would show a transcriptionalresponse of many of the 15 genes previously associated with copperstress in M. tb. In fact, 11/15 of the genes previously associated withtoxic copper response were identified as having significantly changedtranscript levels between ΔctpV and WT during copper stress. A number ofthese genes (N=7) showed a change in the direction of induction (e.g.,genes upregulated in general copper response were downregulated afterdeletion of CtpV).

In addition to the overlap with the copper stress dataset, eighty-sevenother genes were identified, including functional categories notpreviously associated with copper response, such as mammalian cell entry(“mce”) family proteins, ribosomal proteins, and a number ofmembrane/secreted proteins. While these studies have shown that deletionof ctpV invokes a unique response to environmental copper via increasedintracellular copper levels, it is apparent that deletion of ctpV alsohas other affects on the intracellular environment of the bacterium thatcannot easily be attributed to copper concentration alone. Though notwishing to be bound by any particular theory, it is possible that thelack of a membrane protein affecting membrane stability or signaling, ordeletion causes indirect affects on other proteins such as regulatorsresponsive to generalized cellular stress.

The whole-genome microarray of ΔctpV relative to WT at high copper didnot reveal induction of any of the genes with high sequence similarityto CtpV (see Table 1). The more sensitive technique of qRT-PCR was thenused to more precisely investigate the transcriptional induction of allof the other P-type ATPases in the M. tb genome.

These data revealed that only one other P-type ATPase, ctpG, is inducedin the presence of copper, and is particularly induced in the absence ofctpV, as shown in FIG. 14. Referring to FIG. 14, a qRT-PCR survey of theresponse of all predicated metal-transporting P-type ATPases within theM. tb H37Rv genome to copper was conducted. The transcriptional profileat 500 μM copper in the wildtype strain (gray bars) shows induction ofonly ctpG and ctpV. In the absence of ctpV (black bars), the inductionof ctpG is increased. Data are displayed as fold-change relative toexpression at 0 μM Cu, and are normalized to expression of 16S.

A predicted metalloregulatory protein, rv1994c, lies upstream of ctpG,and was identified via microarrays as responsive to general copperstress as well as the absence of ctpV.

EXAMPLE II rv0348 Knockout Mutant

A. Overview

Earlier analysis of the chronic stage of tuberculosis in mice identifiedseveral unique genes induced during chronic infection, including a noveltranscriptional regulator encoded by the rv0348 gene (also known as the“mosR” gene). Transcripts for rv0348 were upregulated ˜200 fold after 60and 140 days of M. tb infection in mice, and the Rv0348 protein wasshown to bind to its own promoter. In this Example, an M. tb mutant isgenerated with an inactivated rv0348 gene to examine the role of thisgene in M. tb survival in the murine model following aerosol infection.

B. The Δv0348 Knockout Mutant Shows Attenuated Activity Compared to itsWild-Type Counterpart

In BALB/c mice, the bacilli load of the Δrv0348 mutant strain wassignificantly lower compared to the H37Rv and rv0348-complementedstrains, indicating a role for rv0348 in controlling mycobacterialvirulence.

1. Strains, Media, Plasmids and Statistical Methods

Escherichia coli DH5a and HB101 were used as host cells for cloningpurposes in all experiments of this Example. M. tb H37Rv and M.smegmatismc²155 strains were grown in 7H9 Middlebrook liquid medium (BDBiosciences, Rockville, Md.) and on 7H10 Middlebrook plates supplementedwith albumin dextrose catalase (“ADC”) and antibiotics, when needed(25m/mlkanamycin or 50 μg/ml hygromycin). Protocols for DNAmanipulations employed throughout this Example, including PCR, cloning,DNA ligations, and electroporation were performed as described in“Molecular cloning, a laboratory manual,” Sambrook, et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y (1989) or according tomanufacturer's recommendations. A list of plasmids and constructs usedin this Example is presented in Table 4. Student's T-test implemented inMicrosoft Excel was used to asses significance difference among samplesat p<0.05 level for the reporter assays and bacterial load counts.

TABLE 4 Plasmids and Constructs used in Experiments of Example 2 PlasmidCharacteristics pYUB854 Cosmid for disruption construct pMV361Integrative mycobacterial shuttle vector. Kan^(R) M. smeg:: RecombinantM. smegmatis harboring the pML21 whole Rv0348 operon under the controlof its own promoter in addition to hsp60. Kan^(R) pML23 pMV361 harboringRv0348 gene. Kan^(R) pCV77 Replicative shuttle vector with promoterlessLacZ transcriptional fusion. Kan^(R) pML24 pCV77 with hygromycincassette in opposite direction to LacZ. Kan^(R), Hyg^(R) pML25 pML24harboring the promoter region of Rv0347 operon. Kan^(R), Hyg^(R) pML26pML24 harboring the promoter region of Rv0167 (mce1 operon). Kan^(R),Hyg^(R) pML27 pML24 harboring the promoter region of Rv0700 (rpsJ).Kan^(R), Hyg^(R) pML28 pML24 harboring the promoter region of Rv3130c(tgs1). Kan^(R), Hyg^(R) pML29 pML24 harboring the promoter region ofhsp60. Kan^(R), Hyg^(R)

The procedures for cloning, overexpression, and purification of M. tbRv0348 in E. coli are described in Talaat et al., 2007, J.Bacteriol.,189:4265-4274. Total RNA samples were extracted from mycobacterialcultures grown to OD₆₀₀=0.5 or 1.5 using Trizol (Invitrogen, Carlsbad,Calif.) as described in Talaat et al., Proc. Natl. Acad. Sci U.S.A,2004, 101:4602-4607 and Talaat et al., 2007, J. Bacteriol.,189:4265-4274. Extracted mycobacterial total RNA samples were treatedwith DNAse I (Ambion, Austin, Tex.) until no DNA was detected using PCRprimers for the 16S rRNA gene. Primers used in Example 2 are listed inTable 5.

TABLE 5 Primers used in Experiments of Example 2 Gene ID sequencepurpose Rv3130c F1 5′-gggtttctcaaggcagaaga-3′ qRT-PCR Rv3130c R15′-ggatcgtccacccatttg-3′ qRT-PCR Rv2628 F 5′-aatccgccaccatctatcag-3′gRT-PCR Rv2628 R 5′-atctcaacggacaggtgctc-3′ gRT-PCR Rv0700 F15′-ggacagaagatccgcatcag-3′ qRT-PCR Rv0700 R1 5′-cccgcgagtccttgtactta-3′gRT-PCR Rv0167 F1 5′-attctttcgcatgtgtgtgc-3′ qRT-PCR Rv0167 R15′-gaagatcaacagcaccgtca-3′ qRT-PCR Rv0347 F1 5′-agcttgccgatctcaaactc-3′qRT-PCR Rv0347 R1 5′-cttctgccggaggttctttc-3 gRT-PCR Rv0569 F5′-cgatagatcaaccggaccac-3′ gRT-PCR Rv0569 R 5′-cattctgctcctccgcagt-3′gRT-PCR Rv1996 F 5′-caacaaacgaacctcggaat-3′ qRT-PCR Rv1996 R5′-tactcaaatgcccacccttc-3′ qRT-PCR Rv1997 F 5′-tcaagaatccaaggcagagg-3′qRT-PCR Rv1997 R 5′-tgactcgttcacgctcaatc-3′ qRT-PCR Rv2032 F5′-gacttggtggagtcgcagtt-3′ qRT-PCR Rv2032 R 5′-ccaatgaactgtgcggtatg-3′gRT-PCR Rv3128c F 5′-gggctcaaagcttctgtcac-3′ gRT-PCR Rv3128c R5′-tggtggcctagtggtttttc-3′ qRT-PCR Rv0348A5′-actagtctacccgggctgggaggagtttcg-3′ Knockout of Rv0348 Rv0348B5′-aagcttgcaaagccgtagtccgcgagctgc-3′ Knockout of Rv0348 Rv0348C5′-tctagatggcgggacatcgcacgcgttgtc-3′ Knockout of Rv0348 Rv0348D5′-ggtaccaacgggccaacggtgctgtcggag-3′ Knockout of Rv0348 Rv0348 F15′-TCGCGGACTACGGCTTTG-3′ qRT-PCR in stress conditions Rv0348 R15′-CCTTGCGCCATTTGGTGATTG-3′ qRT-PCR in stress conditions Rv0348 F25′-atcctctagaatgaccatttcgttct-3′ Cloning of Rv0348 gene in pMAL-c2Rv0348 R2 5′-gcgcaagcttaccgcttgggtcttat-3′Cloning of Rv0348 gene in pMAL-c3 Rv0348 F35′-AAGAATTCGTGCCCGGCGCGCGCGAGTTGACG-3′ Cloning of Rv0348 operon intopMV361 Rv0348 R3 5′-CACCCCGCTCAAGCTTGCCTCGAC-3′Cloning of Rv0348 operon into pMV361 Rv0348 F45′-ggggaattcatgaccatttcgttctctagc-3′ Cloning of Rv0348 gene into pMV361Rv0348 R4 5′-TGGAAGCTTTTACCGCTTGGGTCTTATCGA-3′.Cloning of Rv0348 gene into pMV361 Rv0347 F25′-cggtctagaAttgagctccctgggatggtg-3′ Cloning into pML24 Rv0347 R25′-cggaagcttggccgtcacaacattcatgataa-3′ Cloning into pML24 Rv3130c F25′-cggaagcttGTAACCGCTGCCCGAAC-3′ Cloning into pML24 Rv3130c R25′-cgcggatccCACACCACAGCTGAGGATCA-3′ Cloning into pML24 Rv0700 F25′-cggtctagaCGGGAAGCTCGCAGGTgg-3′ Cloning into pML24 Rv0700 R25′-cggaagcttCTCCCGCGAGTCCTTGTac-3′ Cloning into pML24 Rv0167 F25′-cggtctagaCGAAGACCTAGGTGAGTTCCTG-3′ Cloning into pML24 Rv0167 R25′-cggaagcttGAGCGTGAAGATCAACAGCA-3′ Cloning into pML24 hsp605′-cgctctagacgggtcttgttgtcgttggcgg-3′ Cloning into pML24 hsp605′-cggaagcttcattgcgaagtgattcctccgg-3′ Cloning into pML24 Rv0347 F35′-ttgtcgtgccgaccgtcgcggg-3′ EMSA Rv0347 R3 5′-ggagtccatcgcgccagctcct-3′EMSA Rv0165c F 5′-tcaacggcagcaccacgtgg-3′ EMSA Rv0165c R5′-tgacccgatcgccgaaaccg-3′ EMSA Rv0823c F 5′-acacagcgcccggaatgcga-3′EMSA Rv0823c R 5′-ggaagcccgtacgggcaaga-3′ EMSA Rv1846c F5′-gtgtaggcaaggtcgcggcg-3′ EMSA Rv1846c R5′-ggctgcacgtccttgtgtctacacc-3′ EMSA Rv2160c F5′-cagctcgaacgcgagttggc-3′ EMSA Rv2160c R 5′-aagccatgcctagcgccgac-3′EMSA Rv1996 F 5′-gaagacgaggagcaccggcgct-3′ EMSA Rv1996 R5′-gtgcgcttgggcgaccaggtac-3′ EMSA Rv3139 F 5′-TGCCCAGGCTGCCGGGCAACG-3′EMSA Rv3139 R 5′-gcgcagtgatcggttcagcgga-3′ EMSA Rv0145 F5′-GTCTCTTCGTTGGCCGAGACGCTGT-3′ EMSA Rv0145 R acccgccgacgacaccaacaccEMSA Rv3825c F 5′-ccacttgcacaccgtccgaccg-3′ EMSA Rv3825C R5′-gaagcgtcagactaccggcccg-3′ EMSA Rv3130c_F3 5′-GTAACCGCTGCCCGAAC-3′EMSA Rv3130c_R3 5′-CACACCACAGCTGAGGATCA-3′ EMSA Rv0700_F35′-CGGGAAGCTCGCAGGT-3′ EMSA Rv0700_R3 5′-CTCCCGCGAGTCCTTGT-3′ EMSARv0167_F3 5′-CGAAGACCTAGGTGAGTTCCTG-3′ EMSA Rv0167_R35′-GAGCGTGAAGATCAACAG CA-3′ EMSA

2. Construction of the rv0348 Knockout Mutant

The strategy to construct the rv0348 mutant included the insertion of ahygromycin cassette within the coding sequence of the rv0348 gene usinga specialized transduction-based protocol. Attempts to delete the wholegene with the 200 by flanking sequences failed to yield any mutants.Earlier transposon mutagenesis indicated the rv0347 gene flanking therv0348 sequence was essential, explaining the failure to delete thewhole rv0348 gene where flanking sequences were disrupted. However,following specialized transduction of the insertion constructs(introducing the hygr sequence at 269 by after the start of rv0348) tothe M. tb H37Rv strain, several transductants were obtained. The codingsequence of rv0348 could not be replaced by hygR sequence so thegenerated mutant had all the sequence of rv0348 but with hygR insertedat 269 by after the translation start of rv0348.

A specialized transduction protocol was adopted with a few modificationsto inactivate the rv0348 gene using the virulent strain of M. tb H37Rv.Approximately 800 bp-fragments flanking the rv0348 ORF (specifically,flanking the 269 bp) were amplified using standard PCR protocols.Amplicons were cloned into pGEM-T vector (Promega, Madison, Wis.) andsequence verified before ligation into the pYUB845 vector using Spel andHindIII for left arm and Xbal and Acc65I for right arm to form theAllelic Exchange Substrate (“AES”). Construction of specializedtransducing mycobacteriophages and transduction protocols were performedas described in Bardarov et.al., 2002, Microbiol., 148:3007-3017.Following 6 weeks of incubation at 37° C., hygromycin-resistant colonieswere selected for further analysis. PCR and Southern blot analyses wereused to verify the mutant genotypes as described before (see, Talaat, etal., 2000, Am. J. Vet. Res., 61:125-128 and Wu et al., 2007, J.Bacteriol., 189:7877-7886). PCR, sequencing and Southern blot analysesof several transductants, shown in FIG. 16, verified the desiredgenotype (Δrv0348) in all transductants, and one of them was chosen forthe rest of the analyses.

FIG. 15 shows the organization of the rv0348 operon and the strategy forgene disruption. Data in FIG. 16(B) show a Southern blot analysis ofSa/I-digested genomic DNA of the H37Rv WT and Δrv0348/ Δrv0348 mutant.Data in FIG. 16(C) show PCR analysis of cDNA synthesized from RNAsamples purified from H37Rv (lanes 1, 3, 5) or Δrv0348/ Δrv0348 mutant(lanes 2, 4, 6). Presence of transcripts of the upstream and downstreamgenes of the rv0348 gene indicate that the Δrv0348 mutant is non-polar.

To generate antibodies against purified Rv0348, two adult maleNew-Zealand White rabbits were inoculated with 125 μg of the recombinantfusion protein in Fruend's incomplete adjuvant (Sigma, St. Louis, Mo.)using an approved protocol by the Institutional Animal Care and UseCommittee. Rabbits were housed individually in cages at 15 to 18° C. andgiven antibiotic-free food and water ad libdium. Each immunization wasadministered subcutaneously (12.5 μg), intradermally (37.5 μg),intramuscularly (50 μg), and intraperitoneally (25 μg) in accordancewith the manufacturer's suggestions. Injections of the antigen-adjuvantmixture were administered every 3 weeks for a total of threeimmunizations. Antibody titers for seroconverted rabbits were measuredby ELISA and immunoblot using recombinant purification tag-specificantibodies.

For immunoblotting, mycobacterial cultures were harvested and lysed byboiling in PBS buffer. Total crude extracts were centrifuged and solublelysates and insoluble pellets were separated on 12% SDS-PAGE andtransferred onto PVDF membrane (Hybond-P, Amersham Biosciences).Membranes were saturated by 5% dried milk and rabbit polyclonal antibodywas used as primary antibody at a dilution of 1/5000 for 2 hrs. Horseraddish peroxidase conjugated to goat anti-rabbit IgG (Pierce ThermoScientific, Rockford, Ill.) was used as secondary antibody at 1/30000.Membranes were developed by Chemiluminescent kit according to themanufacturer's protocol (Pierce).

Data in FIG. 16(D) show a Western blot analysis for different M. tbstrains using polyclonal antibodies raised in rabbits against MBP-Rv0348protein. Pellets from (1) M. tb H37Rv, (2)41-v0348 mutant, (3)Δrv0348::rv0348 complemented strain, and (4) H37Rv::rv0348overexpression strain, were subjected to immunoblotting. The level ofexpression of Rv0348 protein in M. tb H37Rv compared to other constructswas examined in order to determine its expression (or lack ofexpression) in M. tb bacilli with variable rv0348 constructs. Rv0348 wasdetectable, but at low levels, when mycobacterial pellets not culturefiltrate samples were analyzed using Western blot, indicatingintracellular expression of the Rv0348 protein. The blot for solublefractions was negative (data not shown).

3. Construction and Evaluation of an rv0348 Complement

The selected Δrv0348 mutant was further used to construct thecomplementation strain where the coding sequence of rv0348 is expressedin-trans using pMV361-rv0348 to yield the Δrv0348::rv0348 construct.Attempts to introduce the whole operon into M. tb were unsuccessful dueto several genomic rearrangements (data not shown). As can be seen inFIG. 15, growth curves for all strains—wild type H37Rv, its isogenicmutant Δrv0348, complemented strain Δrv0348::xv0348, and M. tb H37Rv:rv0348—showed no measurable difference during in vitro growth inMiddlebrook 7H9 broth. The four M. tb strains were inoculated intoMiddlebrook 7H9 broth at O.D600 0.02 and cultures were shaken at 37° C.in an incubator for six days. OD was monitored during the incubationtime.

For complementation experiments, the coding sequence of the entirerv0348 operon (2.3 kb) or the coding sequence of the rv0348 gene alone(-654 bp) were amplified by PCR. Amplicons were cloned into pGEM-Tvector and subsequently verified by DNA sequencing. Vectors were doubledigested by EcoRI and HindlIl restriction enzymes followed by ligatinggel-purified inserts into pMV361 to give rise to pML21 (Oprv0348) andpML23 (rv0348) shuttle vectors for the expression of the whole operon orrv0348 gene, respectively. Both plasmids (pML21 and pML23) wereindependently electroporated into electrocompetent M. smegmatis and M.tb H37Rv cells. Transformants were selected and subsequently analyzed byPCR to verify integration of the delivered sequences into the M. tbgenome. Expression of the Rv0348 protein was further examined usingimmuno-blotting as described above.

4. Evaluation of Δrv0348, Complement Δrv0348:rv0348 and Wild-Type M. tbstrain H37Rv

Three groups (N=40) of 5-week old BALB/c mice were infected with H37Rv,Δrv0348 or Δrv0348:: rv0348 strains and housed in BSL3 environment usingan approved protocol from the University of Wisconsin, InstitutionalAnimal Care and Use Committee (“IACUC”). Cultures of M. tb H37Rv,Δrv0348 and Δrv0348:: rv0348 strains were grown to mid-log phase(OD₆₀₀=1). A total of 10 ml cultures adjusted to OD₆₀₀=0.3 weresuspended in sterile PBS buffer and used for aerosolization using theGlass-Col aerosol chamber (Terra-Haute, Ind.) to generate an infectiousdose of 200-400 cfu per mouse. Two mice were sacrificed at 4-6 hourspost infection to enumerate the infectious dose by plating on 7H10Middlebrook plates in the presence of hygromycin and/or kanamycin forthe mutant and complemented strains, respectively. Mice (N=5) weresacrificed at different times post infection to remove lungs for platingon Middlebrook 7H 10 agar for colony counting. Portions of livers,spleens and lungs were fixed in 10% neutral buffered formalin for atleast 2 hrs before sectioning and staining with hematoxylin and eosin(“H&E”) stain and Ziehl-Neelsen stain.

Culturing lung tissues at day 1 post infection confirmed that all groupsof mice received a comparable infectious dose of each strain (220CFU/mouse for H37Rv, 350 CFU/mouse for the Δrv0348 and, 460 CFU/mousefor the Δrv0348:: rv0348). Murine survival and lung colonizationfollowing aerosol infection M. tb with different copies of rv0348 isshown in FIGS. 18-20. FIG. 19 shows survival curves of 3 mice groups(N=10) infected with H37Rv, Δrv0348 and Δrv0348::rv0348. FIG. 18 showslung CFU/GM murine lungs following aerosol infection with H37Rv, Δrv0348and Δrv0348::rv0348. The “*” denotes significant difference at thesetimes (Student's t-Test). FIG. 20 shows histological analysis of lungsections of mice lungs at 2 weeks and at 20 weeks after infection withH37Rv, Δrv0348, and Δrv0348::rv0348. Note the level of accumulation ofinflammatory cells (arrow heads) in H&E stained sections of mice groups.

As shown in FIG. 19, all mice infected with the Δrv0348 mutant survivedthe infection up to 40 weeks post infection (“WPI”; end of experiment)with a median survival time (“MST”) >40 weeks, while mice infected withthe wild type strain, H37Rv began to die at 27 WPI with MST=29 weeks. Asshown in FIG. 18, the mycobacterial count for the Δrv0348-infected groupwas significantly different from other groups, especially at 2, 30, 38WPI, as at these WPI, the bacterial loads of the mutant strain weresignificantly lower than the wild type strain. This lower bacterial loadof the mutant strain is further demonstrated by data shown in FIG. 21,which shows CFU/g tissue in murine lungs at time of death for H37Rvwild-type strain, which occurred at 37 weeks, and for Δrv0348 strain,which occurred at 62 weeks.

The difference in CFU counts was mostly observed following 30 WPI andlater, indicating a critical role for rv0348 in the chronic rather thanthe early phase of tuberculosis, where the difference was observable inonly one data point. Initial colony counts of the complemented strain,Δrv0348::rv0348 at 2 WPI were similar to those of the wild type H37Rvstrain indicating successful complementation of Rv0348. Nonetheless,colonization levels at subsequent times, in addition to its survivalcurve, indicated only a partial complementation of Rv0348 activity. PCRanalysis of the retrieved colonies from mice lungs infected with theΔrv0348::rv0348 strain at all times indicted the stability of thecomplementation construct, even after 30 WPI in mice (data not shown).Failure of complementation after 8 weeks of infection could be explainedby the strength of rv0348 operon promoter in comparison to hsp60promoter used in the complementation construct. LacZ assays in M.smegmatis showed that hsp60 promoter is at least 4 times weaker than thepromoter of rv0348 operon (data not shown). Deficiency of in vivocomplementation was encountered for several other genes using similarcomplementation vectors and strategy.

As shown in FIG. 20, histopathological analysis of infected mice tissuesshowed typical, progressive granulomatous lesions in lungs of miceinfected with the wild type or complemented strains where aggregates offoamy macrophages were observed by 20 WPI. Granulomas were more visibleas the disease progressed where they occupied almost the whole lungs bythe time of death (˜29 WPI). Conversely, a lower level of macrophage andlymphocyte aggregates were observed in mice infected with the Δrv0348mutant. Notably, inflammatory lesions were visible in 100% of lungsections of mice infected with H37Rv starting at 20 WPI and later, whilelesions were observed in only 50% of lung sections of mice infected withthe Δrv0348 mutant. This level of inflammatory response did not changesignificantly by the end of the experiment (40 WPI) whenΔrv0348-infected mice were sacrificed. Overall, bacterial loads andsurvival curves in addition to histopathological analyses indicated theattenuation of the Δrv0348 mutant, especially during chronictuberculosis.

C. rv0348 Function under Variable Stress Conditions

The transcriptional profiles of the isogenic mutant relative to itsparental strain, H37Rv, indicated the regulation of several gene groupsorganized into operons and regulons including those involved inmammalian cell entry (mcel), hypoxia (dosR) and starvation.

The ability of the Rv0348 protein to affect transcription of these geneswas further analyzed using a LacZ reporter assay and quantitative,real-time PCR (qRT-PCR).

1. Expression of rv0348 under Variable Stress Conditions

Transcripts constituting the rv0348 operon are known to be activatedduring starvation and extended anaerobic conditions. To examine the roleof rv0348 gene in other mycobacterial defenses, cultures of the wildtype strain H37Rv and its isogenic mutant, Δrv0348, were exposed tostress conditions that are thought to be activated during intracellularsurvival of M. tb. Following exposure to variable stressors, both colonycounts and transcriptional profiles of rv0348 transcripts were assayed(for the H37Rv strain only).

Cultures for M. tb H37Rv, H37RvΔrv0348, complemented strain, Δrv0348::rv0348, or H37Rv M.tb:rv0348 were grown to early log phase (OD₆₀₀=0.5)and their colony counts were determined by plating on Middlebrook 7H10agar plates in order to calculate the viable cells at the beginning ofthe experiment. Aliquots (10 ml) were subjected to 0.05% SDS treatment(Sodium Dodecyl Sulfate, Sigma) for 4 hrs at 37° C. or to heat shock at45° C. for 24 hrs in a slow-shaking incubator. To test static growthconditions, 50 ml-aliquots of M. tb constructs were allowed to grow for2 and 6 months without shaking in closed Falcon tubes. At the designatedtimes, culture aliquots were plated and counted on Middlebrook 7H10agar. Other aliquots were used for RNA isolation to assess theexpression of rv0348 under the examined stress conditions usingquantitative, real-time PCR (“qRT-PCR”).

For qRT-PCR, cDNA was synthesized from 1 μg of total RNA usingSuperScript III (Invitrogen) as directed by the manufacturer, in thepresence of SYBR green and 250 ng of mycobacterial genome-directedprimers. SYBR green qRT-PCR was done using gene specific primers (Table5) at a concentration of 200 nm. The thermocycle conditions were: 95° C.for 3 min, and 40 cycles of 95° C. for 15 S and 60° C. for 30 S. qRT-PCRreactions were performed in triplicates and the threshold cycle valueswere normalized to levels of 16SrRNA transcripts and fold changes werecalculated by ΔΔC_(T) method.

Transcriptional Analysis was performed as follows. Before DNA microarrayhybridizations, double-stranded cDNA (ds-cDNA) was synthesized from 10ug of total RNA using the SuperScript Double-Stranded cDNA Synthesis Kit(Invitrogen) as directed by the manufacturer, in the presence of 250 nggenome-directed primers. The ds-cDNA was cleaned up and labeledfollowing the NimbleGen gene expression analysis protocol (NimbleGenSystems, Inc., Madison, Wis.) and hybridized to NimbelGen-manufacturedmicroarrays following a protocol we established earlier. In thismicroarray, each of the 3989 open reading frames (“ORFs”) encoded in thegenome of M. tb H37Rv strain, was represented by nineteen of 60meroligonucleotide probes. Further, the whole genome was represented fivetimes on each chip (i.e. 5 technical replicates/chip) for a total of 95probes/gene. All hybridizations (3 μg of double-stranded cDNA/Chip) wereperformed using NimblGen hybridization buffer and commercialhybridization chambers (TeleChem International, Inc., Sunnyvale, Calif.)overnight at 42° C. Following hybridization, washing steps wereperformed using Nimblegen washes I, II, and III as recommended by themanufacturer. Slides were scanned using an Axon GenePix 4000B scanner(Molecular Devices Corporation, Sunnyvale, Calif.) and fluorescentintensity levels extracted using NimbleScan (NimbleGen) and normalizedto a mean value of 1,000. Determination of significantly changed geneswas performed using a flexible empirical Bayes model; specifically, theLNN model in the EBArrays package employing an R language (R is an opensource platform used by Bioconductor, an open source and opendevelopment software project). A cutoff of 0.50 for the probability ofdifferential expression (PDE>0.5) was used to determine significantlychanged genes. Statistical enrichment of gene groups within themicroarray genes vs. other transcriptoms were calculated using astandard hypergeometric distribution function in Microsoft Excel.

FIG. 22 shows the transcriptional profile of rv0348 in M. tb H37Rv byqRT-PCR of rv0348 transcripts under variable stress conditions, such ashigh temperature (45° C.), H₂O₂ ₍10 mM) and SDS (0.05%) treatments, aswell as transition to stationary phase (OD₆₀₀=1.5). Fold change wascalculated relative to transcripts in untreated cultures of M. tb H37Rvstrain (OD₆₀₀=0.5). Error bars represent±standard deviations from themeans (bars). qRT-PCR showed that the transcripts of rv0348 remainedunchanged when M. tb cultures were exposed to SDS (0.05%).

The transcriptional profile of rv0348 indicated induction at 45° C. andrepression following exposure to high levels of H₂O₂ or during thestationary phase of growth. On the other hand, no difference in colonycounts was found when mycobacterial cultures (H37Rv and Δrv0348) wereexposed to any of the examined stressors including culturing understatic conditions for 2-6 months at 37° C. As shown in FIG. 23,different M. tb strains were grown in Middlebrook 7H9 liquid medium toearly log phase (OD600=0.5) and their counts were determined. Aliquotsof each strains (10 ml) were subjected to different stressor (SDS 0.05%,for 4 hrs) or to heat shock at 45° C. for 24 hours and the CFUs weredetermined for all strains by plating on Middlebrook 7H10 plates at 37°C.

2. Global Changes in M. tb Transcriptome Triggered by rv0348

It has been suggested that rv0348 plays a role in the transcriptionalregulation of M. tb during the transition to the chronic stage in murinelungs. To identify genes under control of rv0348, cultures of both H37Rvand its isogenic mutant, Δrv0348, were grown in vitro for DNA microarrayanalysis using a high-sensitivity oligonucleotide microarray platform.

Replicate microarray hybridizations were performed for at least twobiological samples of both the wild type and mutant strains and showed ahigh correlation level (r=0.9). Using a standard protocol for Bayesianstatistics, significantly regulated genes with a probability ofdifferential expression (PDE) >0.5 and >±2 fold change between H37Rv andΔrv0348 mutant were identified. Using these criteria, a significantchange in a set of 163 genes (Table 11) was identified between thetranscriptomes for H37Rv and the Δrv0348 (rv0348-regulon).

Table 11 shows a list of the 163 genes whose expression differs inΔrv0348 as compared to the wild-type M. tb counterpart H37Rv. Functionalcategory code for Table 11: 0 virulence, detoxification, adaptation; 1lipid metabolism; 2 information pathways; 3 cell wall and cell;processes; 4 stable RNAs; 5 insertion seqs and phages; 6 PE/PPE; 7intermediary metabolism and respiration; 8 unknown; 9 regulatoryproteins; 10 conserved hypotheticals; 16 conserved hypotheticals with anorthologue in M. bovis. A partial list of significantly changed genesorganized into operons, as determined by an operon prediction algorithm,is listed in Table 6.

TABLE 6 A List of Mycobacterial Operons under Positive and NegativeControl of Rv0348 No. Gene-ID Operon Name* Putative Function PositiveRegulation 1 Rv0167-0177 mce1 Mammalian cell entry operon 2 Rv0684-0685fusA-tuf Elongation factor 3 Rv0700-0710 rpsJ-rpsQ 30S ribosomal proteinS10 4 Rv0718-0723 rpsH-rplO 30S ribosomal protein S10 5 Rv1184c-1185cRv1184c-1185c Conserved hypothetical protein, acyl- CoA synthase 6Rv1613-1614 trpA-ltg Tryptophan synthase α chain, prolipoproteindiacylglyceryl transferase 7 Rv2391, 2392 nirA-cysH Probable nitritereductase/sulphite reductase 8 Rv2948c-Rv2950c fadD22-fadD29 acyl-CoAsynthase 9 Rv3148-Rv3154 nuoD-nuoJ NADH dehydrogenase chain D-J 10Rv3460c** rpsM-J 30S ribosomal protein S13-L36 11 Rv3824c-Rv3825cpapA1-pks2 PKS-associated protein, unknown function, polyketide synthase12 Rv3921c-Rv3924c rnpA, rpmH Unknown membrane protein NegativeRegulation 13 Rv0823c-Rv0824c desA1 Transcriptional regulator, ntrB(NifR3/Smm1 family) 14 Rv1622c, Rv1623c cydB, appC Cytochrome dubiquinol oxidase subunit II 16 Rv2031c** hspX 14 kD antigen, heat shockprotein Hsp20 family 17 Rv2629-Rv2630 Rv2629-Rv2630 Hypothetical protein18 Rv3048c** nrdG Ribonucleoside-diphosphate small subunit 19 Rv3053c**nrdH Glutaredoxin electron transport component of NrdEF 20 Rv3139-Rv3140fadE24, fadE23 acyl-CoA dehydrogenase *Operon predications are based onearlier analysis. **Single genes of a larger operon or regulon.

Induced genes in the H37Rv transcriptome compared to the Δrv0348 mutant(N=98 genes) are suggested to be under the positive control of therv0348 while repressed genes (N=65) are suggested to be under itsnegative control. A representative sample of genes that showedtranscriptional changes by DNA microarray analysis was verified byqRT-PCR. In all of the examined genes (N=10), there was an agreement ofthe transcriptional change (either induction or repression) between DNAmicroarray and qRT-PCR analyses, as shown in Table 7 and FIG. 24.

TABLE 7 Confirmation of Microarrays Data with Real-Time qRT-PCR qRT-PCRqRT-PCR Δrv0348/ Microarrays Δrv0348::rv0348/ Genes WT SD Δrv0348/WT PDEWT SD Rv0167 −7.69 0.7 −8.9 1.00 −1.38 0.4 Rv0347 54.19 0.9 4.6 1.002.02 0.3 Rv0569 42.99 0.3 6.4 1.00 1.22 1.7 Rv0700 −2.13 0.7 −3.5 1.00−1.03 0.3 Rv1996 32.60 0.4 12.7 1.00 2.89 0.3 Rv1997 565.48 0.3 3.4 1.003.23 0.4 Rv2032 169.29 1.2 3.8 0.99 3.23 0.5 Rv2628 448.82 0.2 15.0 1.004.05 0.4 Rv3128 853.16 0.9 0.0 0.00 4.05 0.4 Rv3130c 8.34 0.8 4.4 0.99−1.61 0.6

FIG. 24 shows fold changes of ten genes utilizing RNA from both mutantΔrv0348 and complemented 41v0348:n70348 strains relative to H37Rv wildtype strain. Both the Δrv0348 and Δrv0348::rv0348 are represented byblack and grey bars, respectively.

Genes involved in survival during stationary and persistent phases(rpoB) of growth as well as those regulating transcription (e.g., rho,rpmE) (TubercuList database) were among genes under positive control ofRv0348. The positively-regulated operons (Table 6) included the mceloperon (rv0167-rv0177), indicating a role for rv0348 in regulatingvirulence of M. tb. Other genes induced in the presence of rv0348included a tryptophan biosynthesis gene (trpA), translation apparatusoperon (fusA-tuf) and the ribosomal biosynthesis operon (rv0700-rv0723)(Table 6). Several other regulatory genes were also among theRv0348-regulon including the hupB (encodes a DNA-binding protein) andrho (transcription termination factor). In E. coli, the expression ofthe tryptophan operon is regulated by inhibition of ribosomal bindingsites. It is noteworthy to mention here that functional orthologues totrp operon regulatory genes are induced by rv0348 (e.g., 50S ribosomaloperon, rho gene) indicating the ability of Rv0348 to exert itsregulatory role(s) through transcriptional inhibition. Furthercomparative analysis to the starvation-induced transcriptome analyzedbefore identified a set of eighteen genes that are positively-regulatedby Rv0348. FIG. 25(A) shows a Venn diagram representing the number ofrv0348-positively regulated genes compared to genes induced undernutrient starvation (Betts et al., 2002, Mol. Microbiol., 43:717-31) andthose repressed in the phagosome environment (Schnappinger, D. et al.,2003, J. Exp. Med., 198:693-704). Among the rv0348-positively regulatedgenes are a group of twenty-nine genes that were repressed duringmacrophage infection. This profile indicates the ability of M. tb tomodulate levels of gene transcripts to survive the macrophageenvironment using a rv0348-dependent mechanism.

Rv0348-negatively regulated genes included a significant number ofphagosome-activated genes (N=33). Among this group is rv3130c whichencodes triglyceride synthase (tgs1), a protein that is involved intriglyceride synthesis in M. tb, indicating a role for rv0348 inregulating mycobacterial fatty acid metabolism. A set of therv0348-negatively regulated genes (N=24) were among the 47 genesresponsive to hypoxia (see, FIG. 25(B)) or to the 48 genes responsive toreactive nitrogen intermediates (“RNI”) and gradual adaptation to lowlevels of oxygen. Additionally, a list of thirty-three genes that wereactivated during anaerobic growth of M. tb were also found among therv0348-negatively regulated genes in this study. FIG. 25(B) shows a Venndiagram representing the number of rv0348-negatively regulated genescompared to genes induced under hypoxia (Park, H. D. et al., 2003, Mol.Microbiol., 48:833-43) and anaerobic conditions (Muttucumaru, D. G. N.et al., 2004, Tuberculosis, 84:239-46). Previously, a significant levelof overlap existed between the hypoxia and RNI regulons and were shownto be under the two-component regulator, dosR. Transcripts for the dosRregulator did not change in the present analysis, indicating anadditional and/or alternative role(s) for the set of twenty-four genesin the pathobiology of M. tb. However, the activation of the acr gene isusually considered a strong indication of the activation of the dosRregulon. The acr gene was previously confirmed to contribute to M. tbsurvival in macrophages, hence its inclusion under negative control ofrv0348 indicates a potential role for rv0348 in down-regulating genesinvolved in hypoxia, in stages when they are not needed. Finally,hypergeometric distribution analysis of the Rv0348-dependent genes andeach of the compared transcriptomes indicated the significantassociation between the rv0348-induced transcriptome and starvation,phagosome survival, hypoxia and anaerobic conditions (p<0.001), as shownin Table 8.

TABLE 8 Analysis of Groups Overrepresented in the Transcriptome ofMycobacterium tuberculosis as Determined by Hypergeometric Distribution# in rv0348 # in Category Transcriptome Conditions P-value Starvation 16114 9.0E−06 Low in Phagosome 27 127 3.2E−13 Hypoxia 24 48 0.0E+00Anaerobic 33 231 6.3E−11

Overall, the presented analyses show the previously undiscovered, yetbroad and far reaching potential regulatory roles exerted by the rv0348in M. tb survival strategies.

3. rv0348 Expression in M. smegmatis Model of Hypoxia

The study of rv0348 expression under hypoxic conditions in M. smeg::pML2lwas performed using the Wayne model of hypoxia in M. tb, which hasproven equally useful in studies of M. smegmatis. Briefly, a singlecolony of M. smeg::pML21 (see, EXAMPLE II section B.3 for construction)harboring the rv0348 operon was grown with shaking at 37° C. to an OD₆₀₀of 1.0 in Dubos Tween Albumin medium (BD Biosciences) supplemented withkanamycin (30 gg/ml). This culture was used to inoculate 6 ×30 mlscrew-capped tubes containing stir bars to an OD₆₀₀ of 0.1 in Dubosmedia containing methylene blue (1.5 μg/ml) to serve as an indicator ofoxygen levels. Three tubes were used as aerobic controls with loosecaps, a head space ratio (“HSR”) of 1.5, and were stirred at 200 rpm.The remaining 3 tubes were used for the anaerobic cultures withtightened, parafilm-sealed caps, an HSR of 0.5, and were stirred at 120rpm. The color of the tubes was monitored and aerobic/anaerobic tubeswere taken for analysis when the methylene blue first showed signs offading (day 1) and after the anaerobic cultures became completelycolorless (day 6 and 7). An aliquot (100 μl) of each sample was platedfor colony counts while the rest was used for total RNA extraction andqRT-PCR as described above. The whole experiment was repeated 3 times.

Using the Wayne model of hypoxia in M. tb, transcripts of rv0348 operonwere modestly induced under anaerobic conditions indicating the rv0348operon's involvement in hypoxic responses. Since large number of thedormancy regulon genes were suggested to be controlled by rv0348, it ispossible that rv0348 could be involved in mycobacterial hypoxicresponses. To test this hypothesis, an in vitro model of hypoxia wherethe influence of hypoxia and anaerobic conditions on rv0348 operon couldbe studied in a recombinant strain M. smegmatis (M. smeg::pML21) wasdeveloped that was shown to express Rv0348, as shown in FIG. 20(A). FIG.20 shows the transcriptional regulation of Rv0348-dependent genes inpresence or absence of the rv0348 operon. Referring to FIG. 20, the *denotes significant change in a Student's t-Test (p<0.001). FIG. 20(A)shows a Western blot analysis of the recombinant strain of M. smegmatismc²155 expressing Rv0348 protein. FIG. 20(B) shows the survival curve ofM. smeg::pML21 under aerobic and anaerobic conditions (left scale) andfold change in rv0348 transcripts as measured by qRT-PCR (right scale).

Construction of LacZ vectors and β-galactosidase assays were performedas follows. The DNA fragment corresponding to the putative promoterregions of rv3130c, rv0167, rv0700, rv0347 and hsp60 genes were clonedby PCR using gene-specific primers (Table 5). The different promoterswere cloned into pML24 shuttle vector (a derivative of pCV77 vectorwhere a hygromycin cassette was cloned into a Spel site). M. smegmatiswas first electroporated by pML21 and positive clones were selected andverified by PCR and Western blot to ensure the expression of Rv0348protein. Recombinant M. smegmatis were electroporated with a differentshuttle vector (pML24 derivative) and incubated on selective LB platessupplemented with 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside(X-Gal). All recombinant M. smegmatis developed blue color on platesexcept the negative control (pML24). Assessment of β-galactosidaseactivity in different constructs was performed in sonicated extracts ofM. smegmatis strains using a β-galactosidase assay kit (Stratagene,Cedar Creek, Tex/) according to the manufacturer protocol. Experimentswere carried out in triplicate and repeated twice from independentcultures with different amounts of soluble fraction proteins.β-galactosidase was expressed as Miller units/mg of soluble lysate. FIG.26(C) shows Lac-Z repression for constructs for rv3130c promoter. FIG.26(D) shows Lac-Z repression for constructs for rv0347. FIG. 26(E) showsLac-Z repression for constructs of rv0700. FIG. 26(F) shows Lac-Zinduction for constructs of rv0167.

BLAST analysis indicated that the rv0348 operon is absent from thegenome of M. smegmatis, allowing for the expression of Rv0348 andassessment of its function(s) in the rapidly growing M. smegmatis. Usinga modified version of the Wayne model of hypoxia, it was found thataerobic cultures of M. smeg::pML21 grew to a higher density than theanaerobic cultures, as expected (see, FIG. 26(B)). Interestingly,transcripts of rv0348 were significantly up-regulated in the culturesgrown under hypoxic conditions (at day 1) with a more profound inductionwhen cultures reach anaerobic phase by day 6 of incubation. Thisdramatic increase in rv0348 transcripts strongly supports the hypothesisthat rv0348 participates in M. tb response to anaerobic stress, inaddition to its role in M. tb survival during infection. Currently,experiments are underway to examine the survival of H37RvΔrv0348 mutantunder anaerobic environment.

4. Rv0348 binding to New Promoter Regions

It has been shown that rv0348 encodes a transcriptional regulator thatbinds to its own promoter. Electrophoresis mobility shift assay (“EMSA”)assays were performed using the predicted regulatory sequences of tengenes that changed their transcriptional profile based onpresence/absence of intact rv0348 gene to examine the ability of Rv0348to regulate other genes. For EMSA assays, the Rv0348 protein waspurified as detailed in Talaat et al., 2007, J. Bacteriol.189:4265-4274. Probes were generated using standard PCR amplificationprotocols and primers designed by Primer3 v. 0.4.0 by providing theupstream probable regulatory sequences of selected genes. Potentialpromoter regions of several selected genes were amplified by PCR andend-labelled by radioactive P32. The different probes were allowed tobind to recombinant MBP-Rv0348 and subsequently run on 4% nativepolyacrylamide gel. The gel was then dried and exposed to Kodak film. Asshown in FIG. 27, the presence of Rv0348 did not impact the migrationpattern of DNA fragments representing any of the ten putative promotersindicating an indirect regulatory function for Rv0348. Only when thepositive control was used (upstream region of the rv0348 operon), aretardation of the DNA migration was noticeable. It is possible thatother regulatory elements are needed to amplify the regulatory role(s)of rv0348.

5. Rv0348 Regulatory Functions

Because of the lack of a direct binding of Rv0348 to any of the examinedgenes with differential gene expression profile, the LacZ reporter genewas employed to examine the regulatory role of Rv0348. For this purpose,the generated M. smeg::rv0348 (M. smeg::pML21) construct was used toexamine the transcriptional regulation of a selected list of genes thatbelong to rv0348-regulon. A verified clone of the M. smeg::rv0348 waselectroporated with derivatives of the pML24 plasmid, listed in Table 4,where the putative promoter regions of several genes were clonedupstream of a promoterless reporter gene (lacZ).

Screening of transformants showed that all constructs formed bluecolonies on plates supplemented with X-Gal except when a promoterlessLac-Z vector was used for transformation (see FIG. 28). Nonetheless,quantitative analysis of β-galactosidase activity of each construct inthe presence/absence of the rv0348 operon showed significant differencesamong constructs depending on presence of the rv0348 operon.

In all examined promoters, a significant change in the expression levelof LacZ was found between constructs where the rv0348 operon was presentcompared to those without. Both the repression of rv0347 (promoter forrv0348) and rv3130c promoters and the induction of rv0167 (promoter formcel) were in agreement with the negative and positive regulation byRv0348, respectively, as indicated by DNA microarrays. However, in thecase of rv0700 (promoter for ribosomal protein operon), the LacZ assayindicated its repression despite evidence that it is under positivecontrol of Rv0348, indicating the presence of other regulatorymechanisms, besides rv0348, that control rv0700 in M. smegmatis.Interestingly, the LacZ reporter technology was able to showdifferential regulation for mcel, rv3130c and rv0700 genes in M.smegmatis expressing Rv0348, despite the inability of Rv0348 to bind totheir putative promoter regions indicating an alternative strategy forgene regulation exerted by Rv0348.

To further confirm the regulatory role of rv0348, qRT-PCR was employedto estimate the transcript levels of regulated genes in the complementedstrain, H37RvΔrv0348::rv0348, compared to the mutant strain H37RvΔrv0348(see FIG. 24). Such analysis was intended to test the ability of rv0348expression in trans to maintain the functional role(s) played by rv0348and provide an additional confirmation of the regulatory role of Rv0348.In all examined genes, the induction/repression levels of transcripts inthe Δrv0348 were consistent with DNA microarray analysis. However,transcripts in the complemented strain for the ten examined genes wererestored to the wild type level (±1) confirming the regulatory role forthe rv0348 gene and the success of the complementation for in vitrocultures. Overall, both the reporter assay and quantitative PCR analysissupported a regulatory role for the Rv0348 as a transcriptional factor.

It has been shown that rv0348 encodes a transcriptional regulator withboth inducer and repressor activities that are used to regulate keymycobacterial responses to stressors such as starvation and low oxygentensions. Based on the presented analyses, a model was generated thatdelineates possible pathways that can be utilized by Rv0348 to explainits role in establishing chronic tuberculosis. Though not wishing to bebound by any particular theory, in this model, as depicted in Scheme 1(a diagram depicting several scenarios in which rv0348 can play a rolein M. tb survival strategies), Rv0348 can bind to its own promoter inorder to maintain a low level of expression especially during log phaseculture or under in vitro growth, in general. During certain stressors(e.g., high O⁻ level), the expression of Rv0348 will be even lower (seeFIG. 26(B)) which will relieve its repression of other genes such ashypoxia- and phagosome-responsive genes.

During other stressors (e.g., high temp or in vivo growth), the rv0348is induced (see FIG. 22), most likely through the activity of othertranscriptional regulators (e.g. SigC) which share a transcriptionalbinding sites upstream of rv0348 operon. Such binding could prevent thebinding of Rv0348 to its promoter, and hence, its own expression will beinduced which in turn could activate genes involved in starvation andinvasion among the rv0348-regulon (see, FIG. 25(A)). Under all of thesescenarios (relieve of hypoxia gene repression or induction of starvationgenes), the general outcome of the induction of the rv0348-regulon isthe fitness of M. tb to persist under variable host microenvironments.

EXAMPLE III rv0990c Knockout Mutant

A. Overview

The rv0990c gene is the central gene of an operon of three genes(rv0989c-0991c) located in the iVEGI (see FIG. 1). Bioinformaticsanalysis suggested that rv0990c could be a DNA-binding protein. Toinvestigate the role of rv0990c in the regulatory pathways andpathogenesis of M. tb, the coding sequence of rv0990c was deleted usinga phage-mediated delivery technique. The animal data obtained (e.g., cfucount, histopathology, and survival curves) following aerosol infectionconfirmed the attenuation phenotype of Δrv0990c.

B. Construction of Δrv0990c Knockout Mutant

A knockout mutant of rv0990c, Δrv0990c, was prepared using the virulentM. tb strain H37Rv. The rv0990c gene is 657 base pairs. 490 base pairsof the coding sequence of rv0990c (from 70-560) were deleted from thegenome ofM.tb and replaced by the hygromycin cassette using the samephage delivery technique employed to disrupt the rv0348 gene andgenerate the knockout mutant. Briefly, ˜800 basepair fragments of boththe upstream and downstream portion of the rv0990c gene were amplifiedby PCR and cloned into the pGEM-T Easy vector (Promega, Madison, Wis.).The fragments were then digested at the flanking restriction enzymesites and ligated into pYUB854. The restriction sites used wereSpel/HindIll and XbaI/KpnI for left flank and right flank respectively.

As described for the rv0348 knockout, the pYUB845 construct was used toform specialized transduction mycobateriophages. Transduction protocolswere performed as described in Bardarov et.al., 2002, Microbiol.,148:3007-3017. Following 6 weeks of incubation at 37° C.,hygromycin-resistant colonies were selected for further analysis.

Confirmation of the mutant genotype was performed as describedpreviously for the ctpV and the rv0348 knockouts, using PCR and Southernblot analyses. (See also, Talaat, et al., 2000, Am. J. Vet. Res.61:125-128 and Wu et al., 2007, J. Bacteriol. 189:7877-7886). Primersused to generate and test this mutant are shown in Table 9.

TABLE 9 Gene-Specific Primers for Construction of Δrv0990c Mutant PrimerSequence Purpose AMT533 ATCACTACTAGTGATAGCGTAGCGGAGTCACCRv0990c flank cloning AMT534 ATCACTAAGCTTAGTGACCGGGTCGTTTTGGTRv0990c flank cloning AMT535 ATCACTTCTAGAGGTCCAGTCCGGGCGCAAAARv0990c flank cloning AMT536 ATCACTGGTACCGAACCTTGGCTGCCGGAAGCRv0990c flank cloning AMT926 TGGTGGACCTCGACGACCTGCAGGRv0990c mutant screening AMT899 GTGGACAGCTTGGCCAAGGTCGGCRv0990c mutant screening AMT900 GCACGCTGGGGACTGCTCGAACRv0990c mutant screening AMT885 GGAACTGGCGCAGTTCCTCTGGGGRv0990c mutant screening

C. Construction of the rv0990c Complement

One of rv0990c mutants was electroporated with a copy of rv0990c genecloned under the control of hsp60 promoter in pMV361. The transformantswere verified by PCR for the construct stability M. tb. One of thecomplemented strains was used to infect a group of BALB/c mice toconfirm the observed attenuation phenotype of the mutant.

EXAMPLE IV rv0971c Knockout Mutant

A. Overview

The rv0971c gene is the last gene of an operon of six genes located into the iVEGI of M. tb. (See FIG. 1). Although, the exact function ofrv0971c is largely unknown, it was annotated as a crotonase in theTuberculist, and the operon is believed to play a crucial role in lipidmetabolism (biosynthesis and degradation). The unique location of theoperon in the M. tb pathogenicity island suggested a role inmycobacterial virulence. Attempts to delete the whole operon from thegenome ofM.tb were unsuccessful. Instead, the rv0971c gene was deletedand virulence of the mutant was studied in an animal model.

B. Construction of the rv0971c Knockout Mutant

The rv0971c gene is 810 base pairs. 674 base pairs of the codingsequence of rv0971c (66-740) were deleted and replaced by a hygromycincassette using the same phage delivery technique described above for therv0990v knockout. The mutants were verified by PCR and Southern blottechniques. The restriction sites were SpeI/HindIII and XbaI/KpnI forleft and right arm respectively. Primers used to generate and test thismutant are in Table 10:

TABLE 10 Gene-Specific Primers for Construction of Δrv0971c MutantPrimer Sequence Purpose AMT563 ATCACTACTAGTCAACTCACTGCGGTTACGCCRv0971c flank cloning AMT564 ATCACTAAGCTTATGCTGGCCTTCCTGCAGAARv0971c flank cloning AMT565 ATCACTTCTAGAGCGGTTGTGCGGAGAGTTCARv0971c flank cloning AMT566 ATCACTGGTACCGACTGGATCATCAAGGGCCARv0971c flank cloning AMT885 GGAACTGGCGCAGTTCCTCTGGGGRv0971c mutant screening AMT897 GTTCTCCTCGGTCAGCGTGGTGACRv0971c mutant screening AMT926 TGGTGGACCTCGACGACCTGCAGGRv0971c mutant screening AMT898 AAGATCACCACCACCGCGCGTCRv0971c mutant screening

C. Construction of the rv0971c Complement

One of the verified mutants was electroporated with a functional copy ofrv0971c under hsp60 promotor control, in the integrative shuttle vectorpMV361. The complementation study is in progress.

EXAMPLE V Evaluation of Wild-Type M. tb Strain H37Rv and M. tb KnockoutMutants

BALB/c mice can be infected with an M. tb knockout mutant (e.g.,Δrv0990c and/or Δrv0971c) or corresponding wild-type M. tb using alow-dose aerosolization protocol. Bacterial survival and mouse lungpathology can be measured at short-term as well as long-term time pointsvia the homogenization and plating of infected lung tissue as well asorgan histology. Additionally, mice infected with the M. tb strains canbe monitored over the long-term course of the infection and the survivalof the infected mice can be recorded. p Briefly, BALB/c mice (Harlan,Indianapolis, Ind.) can be infected in a Glas-Col chamber (Glas-Col,LLC, Terra Haute, Ind.) loaded with 10 mL of an M. tb knockout mutantsuch as Δrv0990c and/or Δnrv 0971c,or the corresponding wildt-type M. tbstrain at OD 0.30. Infectious dose of approximately 300 CFU/animal canbe confirmed via a 1-day time point. CFUs can be determined at differenttime points (e.g., 2 weeks, 4 weeks, and 38 weeks, etc.) by homogenizinglung tissue in PBS buffer and plating on Middlebrook 7H10+10% ADC,followed by incubation at 37° C. for one month. Final CFUs can benormalized to the weight of the lung tissue used. Sections of lung,liver, and spleen tissue can be taken and incubated in formalin prior tosectioning and staining with H&E and AFS. Histopathology slides can beexamined and scored by a pathologist not associated with the study.

EXAMPLE VI A0990c Infected Mice

Three groups of mice were infected with A0990c M. tb knockout mutant asdescribe above in Example V. The progression of the disease wasmonitored in the three groups of infected mice by cfu count and survivalcurves. As shown in FIG. 29, a decrease in CFUs of a Δrv0990c knockoutmutant (derived from M. tb strain H37RV) relative to the correspondingWT H37Rv strain was observed at both short-term and long-term timepoints. Referring to FIG. 29, the bacterial load of mice after infectionwith either wild-type (H37Rv) or its isogenic mutant Δrv0990c wasdetermined at 2, 4, 8, 30 and 38 weeks.

Mice infected with Δrv0990c lived longer than mice infected with thewild-type H37Rv strain, resulting in an increase in time to death, asshown in FIG. 30. Referring to FIG. 30, the survival of mouse groupsafter infection with WT or Δrv0990c is shown.

EXAMPLE VII A0971c Infected Mice

Two groups of mice were infected with wild-type and Δrv0971c withaerosol challenge as described above in Example V, and the cfu count andsurvival were monitored in the two groups. As shown in FIG. 29, adecrease in lung CFUs of a Δrv0971c knockout mutant (derived from M. tbstrain H37Rv) relative to the corresponding H37Rv WT strain was observedat both short-term and long-term time points. Referring to FIG. 29, thebacterial load infection with either wild type (H37Rv) or its isogenicmutant Δrv0971c was determined at 2, 4, 8, 30 and 38 weeks.

Mice infected with Δrv0971c lived longer than mice infected with WT, asshown in FIG. 30. Referring to FIG. 30, the survival of mouse groupsafter infection with WT or Δrv0971c is shown.

EXAMPLE VIII Knockout Mutants Used to Generate an Immune Response inMammals and as Vaccines

Live attenuated mutants can be used as vaccines candidates againsttuberculosis. Additionally, genetic vaccines based on the targeted genescan be used to develop a genetic immunization protocol that can elicitprotection against tuberculosis.

In a typical immunization experiment, hosts (e.g. mice or non-humanprimates) will be immunized with the attenuated mutants. At 4 weeks postinfection, sera or organ tissues can be collected from inoculatedanimals to evaluate the generated immune responses. Both humoral andcellular-based assays can be used to evaluate the host responses toimmunization.

Although humoral and cellular assays can estimate the level of immunitygenerated following vaccination, it will not provide estimate of thelevel of protection offered by each vaccine construct. To estimate theprotective power of vaccine candidates, immunized animals can bechallenged by aerosolization of the virulent strain of M. tuberculosis.The readout of such assays includes animal survival curves, the level oforgan colonization with the virulent strain of M. tuberculosis as wellas immunological and histopathological responses elicited by challenge.

A. Vaccination and challenge of guinea pigs: Female Dunkin-Hartleyguinea pigs (350-450 g) free of infection can be used. Four groups often guinea pigs can be immunized with 75 μl of a 5×10⁴ CFU live knockoutmutant formulation subcutaneously in the nape of the neck. Group I canbe be immunized with the ΔctpV mutant; Group II can be be immunized withthe Δrv0348 mutant; Group III can be immunized with the Δrv0990c mutant,and Group IV can be immunized with the Δrv0971c mutant. A control groupsof 10 guinea pigs can be vaccinated with a 5×10⁴ CFU live BCG Pasteurformulation, and a control group of 10 guinea pigs can be injected withsaline. Five weeks after vaccination, all but two guinea pigs (thecontrols) from each group can be challenged aerogenically with a livesuspension of M. tb strain H37Rv to achieve an inhaled retained dose inthe lungs of approximately 300 organisms.

B. Vaccination and challenge of guinea pigs: Female Dunkin-Hartleyguinea pigs (350-450 g) free of infection can be used. Four groups often guinea pigs can be immunized with 75 μl A of a 5×10⁴ CFU liveknockout mutant formulation subcutaneously in the nape of the neck.Group I can be immunized with the ΔctpV mutant; Group II can beimmunized with the Δrv0348 mutant; Group III can be immunized with theΔrv0990c mutant, and Group IV can be immunized with the Δrv0971c mutant.A control groups of 10 guinea pigs can be vaccinated with a 5×10⁴ CFUlive BCG Pasteur formulation, and a control group of 10 guinea pigs canbe injected with saline. Five weeks after vaccination, the guinea pigscan vaccinated as described above, but with 50% of the CFUs. Five weeksafter the second vaccination, all but two guinea pigs (the controls)from each group can be challenged aerogenically with a live suspensionof M. tb strain H37Rv to achieve an inhaled retained dose in the lungsof approximately 300 organisms.

C. Testing for an immune response in vaccinated guinea pigs: Prior toexposure to the infectious M. tb strain H37Rv., a blood sample can betaken from each of the guinea pigs, including vaccinated andsaline-injected, and the presence or absence of antibodies directed toM. tb can be determined by methods known in the art.

D. Post mortem examination of guinea pigs: Guinea pigs can be sacrificedaccording to institutional protocol after 20 weeks. Tissues of interest(e.g., lung, spleen, etc.) can be harvested immediately after death andanalyzed for M. tb colonization.

E. Bacterial enumeration: CFUs can be determined by homogenizing lungtissue in PBS buffer and plating on Middlebrook 7H10+10% ADC, followedby incubation at 37° C. for one month. Final CFUs can be normalized tothe weight of the lung tissue used. Sections of lung, liver, and spleentissue can be taken and incubated in formalin prior to sectioning andstaining with H&E and AFS. Histopathology slides can be examined andscored by a pathologist not associated with the study.

F. Results: Guinea pigs from Groups I-IV and control guinea pigsvaccinated with BCG Pasteur formulation are expected to have developedantibodies directed to M. tb. Additionally, guinea pigs from thesegroups are expected to live longer and have fewer CFUs in organs andtissues tested, post infection, as compared to guinea pigs injected withsaline. Additionally, further challenges of vaccinated guinea pigs withinfectious M. tb strains is expected to result in less sever symptoms.For example, re-challenged guinea pigs are expected to exhibit anincreased post-infection life span and fewer CFUs in lung, liver andspleen than non-vaccinated guinea pigs. Similar results are expectedwith the “boosted” guinea pigs described in section VIII.B.

In contrast, no or very low titer antibodies directed to M. tb areexpected in the saline-injected guinea pigs. Moreover, subjects in thistest group are expected to die due to the M. tb infection at about 38weeks post infection.

EXAMPLE IX Δctp V Infected Mice

Three groups of mice were infected with ΔctpV M. tb knockout mutant asdescribed above in Example V. BALB/c mice (Harlan Laboratories, Inc.,Indianapolis, Ind., USA) were infected in a Glas-Col chamber (Glas-Col,LLC, Terra Haute, Ind., USA) loaded with 10 ml of either ΔctpV orwildtype bacteria at OD₆₀₀ 0.30. Infectious dose of approximately 300CFU/animal was confirmed via a 1-day timepoint. CFUs were determined byhomogenizing lung tissue in PBS buffer and plating on Middlebrook7H10+10% ADC, followed by incubation at 37° C. for one month. For thesurvival curve, animals were monitored daily by animal care staff notassociated with the study. As specified in our animal protocol, micewere sacrificed after being identified by our animal care staff asmorbidly ill, using criteria such as haunched posture, extreme weightloss, and slow or pained movements. Sections of lung, liver, and spleentissue were taken and incubated in formalin prior to sectioning andstaining with hematoxylin and eosin (H&E) and acid-fast staining (AFS).Histopathology slides were examined and scored by a pathologist notassociated with the study. For immunohistochemistry, the primaryantibody, rabbit anti-interferon gamma (Invitrogen), was diluted 1:1000in Van Gogh Yellow antibody diluent (Biocare Medical, Concord, Calif.,USA) and incubated for one hour. Negative control slides received onlydiluent in lieu of antibody. Primary antibody was detected usingbiotinylated goat anti-rabbit IgG secondary antibody (Biocare Medical)and Streptavidin-horseradish peroxidase (Biocare Medical). Staining wasvisualized with DAB+ (diaminobenzidine) (Dako, Glostrup, Denmark) andcounterstained with CAT hematoxylin (Biocare Medical) mixed 1:1 withdistilled water.

The ctpV gene is part of a 29-gene genomic island previously shown to bepreferentially induced in mice relative to in vitro culture (termed thein vivo expressed genomic island, iVEGI). This suggested that ctpV mightplay a role specific to the in vivo lifestyle of Mtb. In addition,experiments showed that CtpV is a copper exporter, and data suggest thatcopper homeostasis in bacteria may play a role in pathogenesis, althoughthis had never been tested in Mtb. To investigate this hypothesis,groups of BALB/c mice (n=30-40) were infected with H37Rv, ΔctpV, orΔctpV::ctpV using a low-dose aerosolization protocol. Bacterial colonycounts from mouse lung tissue over the course of the infection revealedthat, overall, bacterial survival between the three strains over thecourse of infection was similar, and differences in colonization levelsdid not reach statistical significance at any time point (FIG. 37).

FIG. 37 shows bacterial colonization of mouse lungs after aerosolinfection with either wild-type H37Rv, its isogenic mutant ΔctpV, or thecomplemented strain ΔctpV::ctpV. CFUs were determined via homogenizationof lungs from infected mice (N=3-5 per time point) in PBS and plating on7H10+ADC, with hygromycin added in the case of the mutant andcomplemented strain. CFUs were normalized to grams of lung tissuehomogenized. Colonization levels between the three strains did notdisplay a statistically significant difference over the course of theinfection. The experiment was performed twice, with a representativeexperiment shown.

Survival of mouse groups after aerosol infection with H37Rv, ΔctpV, orΔctpV::ctpV is shown in FIG. 38. Survival is displayed as the time frominfection (week 0) until the time declared morbid by animal care staff.Morbid mice were subsequently euthanized, with tuberculosis determinedas the cause of illness via necropsy. A log-rank statistical test wasused to analyze survival of mice groups. Survival of mice infected withH37Rv was significantly different from those infection wth ΔctpV(p-value=0.002), and those infected with survival was ΔctpV::ctpV strain(p-value=0.02). Experiment was performed once with 10 mice/group.

In contrast to the similarity in bacterial load of the mice infectedwith H37Rv, ΔctpV, and ΔctpV:: ctpV, the mice infected with ΔctpV livedsignificantly longer than mice infected with wild type, with 16-weekincrease in time to death of mice infected with ΔctpV versus miceinfected with H37Rv (FIG. 38 shows survival curves of three mice groupsinfected with H37Rv wild-type strain, ΔctpV strain, and A ctpV::ctpVstrain). In fact, the median survival time for mice infected with H37Rvwas 31 weeks, versus 47 weeks for mice infected with ΔctpV and 42 weeksfor mice infected with ΔctpV::ctpV. As determined by a log-rankstatistical test (Ref), survival was significantly different between theH37Rv and ΔctpV infection (p-value=0.002), and survival was alsosignificantly different between the H37Rv and ΔctpV::ctpV infection(p-value=0.02).

FIG. 39 shows histological (A) and immunohistochemisty (B) analysis oflung sections of mice lungs at 8 or 38 weeks post infection withwild-type (“WT”) M. tb and ΔctpV and ΔctpV::ctpV. Hematoxylin and eosin(H&E) stained mouse lung tissue (40× magnification) throughout theinfection show increased lung damage of the mice infected with H37Rv andΔctpV::ctpV relative to mice infected with ΔctpV. FIG. 39A showsrepresentative images at 8 and 38 weeks post-infection. Inset images(1000× magnification) show the Mtb bacilli (arrow heads) which werevisible in lung tissue starting from 8 weeks forward.Immunohistochemisty of infected lung tissue at variable times ofinfection is shown in FIG. 39B. Lung tissue was sectioned and stainedwith antibody for mouse IFN-γ, which appears brown in the images (40×magnification). Inset images show 200× magnification of lesionsdisplaying IFN-γ expression.

Despite carrying similar levels of bacteria throughout the infection,histology staining of the infected mouse tissue revealed consistentlylower levels of tissue damage in mice infected with ΔctpV versus thewild-type and complemented strains. For example, at 8 weekspost-infection, lung tissue from mice infected with ΔctpV displayedgranulomatous inflammation, whereas mice infected with H37Rv displayedmassive granulomatous inflammation with more lymphocytic infiltration(FIG. 39A). By 38 weeks post-infection, granulomas became more developed(presence of giant cells) and occupied almost the whole lungs of miceinfected with H37Rv, compared to only 50% of tissues of mice infectedwith the ΔctpV mutant. Lesions observed in the complemented strain werevery similar to those observed in mice infected with H37Rv strain.Overall, animal survival and histopathological data indicated theattenuation of the ΔctpV mutant compared to other tested strains.

Lung pathology in tuberculosis is thought to be caused mainly by thehost immune response. In order to investigate a possible mechanism forthe decreased lung pathology and increased survival time of miceinfected with ΔctpV relative to H37Rv and ΔctpV:: ctpV, the lungsections were stained with an antibody against mouse interferon- y, akey cytokine known to be highly expressed during tuberculosis infection.As expected, no indication of IFN-γ expression was seen at 2 weeks postinfection, prior to the start of the adaptive immune response (data notshown). However, by 8 weeks post infection, mice infected with H37Rvshow significant IFN-γ expression, localized in areas of lung tissuedamage, yet mice infected with ΔctpV showed only small amounts of IFN-γexpression (FIG. 39B). Mice infected with ΔctpV::ctpV showed anintermediate level of IFN-γ expression. Interestingly, even at the 38week time point where mice infected with ΔctpV display large amounts oftissue damage, there was still little expression of IFN-γ relative tomice infected with H37Rv.

TABLE 11 Foldchange Functional No. Gene_ID Product Function_Class(Mutant/WT) GG_PDE category Positive Regulation 1 Rv0006 gyrA DNA gyrasesubunit A −2.5 0.92 2 2 Rv0007 conserved hypothetical −2.0 0.49 3protein 3 Rv0058 dnaB DNA helicase (contains −2.4 0.88 2 intein) 4Rv0108c hypothetical protein −2.4 0.87 16.6 5 Rv0145 conservedhypothetical −2.7 0.97 10.5 protein 6 Rv0166 fadD5 acyl-CoA synthase−4.2 1.00 1 7 Rv0167 yrbE1A part of mce1 operon −8.9 1.00 0 8 Rv0168yrbE1B part of mce1 operon −2.0 0.50 0 9 Rv0169 mce1 part of mce1operon, cell −5.1 1.00 0 invasion protein 10 Rv0170 mce1B part of mce1operon −7.2 1.00 0 11 Rv0171 mce1C part of mce1 operon −13.3 1.00 0 12Rv0172 mce1D part of mce1 operon −5.0 1.00 0 13 Rv0173 lprK part of mce1operon −3.0 0.99 0 14 Rv0174 mce1F part of mce1 operon −5.0 1.00 0 15Rv0175 conserved hypothetical −2.0 0.55 3 protein (mce1) 16 Rv0176conserved hypothetical −2.2 0.69 3 protein (mce1) 17 Rv0177 conservedhypothetical −4.0 1.00 10.5 protein (mce1) 18 Rv0249c probable membraneanchor −2.2 0.71 7 protein 19 Rv0364 conserved hypothetical −2.2 0.74 3protein 20 Rv0430 hypothetical protein −3.7 1.00 10.5 21 Rv0469 umaA1unknown mycolic acid −2.3 0.52 1 methyltransferase 22 Rv0635 conservedhypothetical −3.1 0.99 10.5 protein 23 Rv0636 hypothetical protein −2.60.85 7 24 Rv0637 conserved hypothetical −2.2 0.69 10.5 protein 25Rv0642c mmaA4 methoxymycolic acid −2.1 0.41 1 synthase 4 26 Rv0667 rpoB[beta] subunit of RNA −2.4 0.91 2 polymerase 27 Rv0684 fusA elongationfactor G −2.7 0.98 2 28 Rv0685 tuf elongation factor EF-Tu −3.7 1.00 229 Rv0700 rpsJ 30S ribosomal protein S10 −3.5 1.00 2 30 Rv0701 rplC 50Sribosomal protein L3 −4.7 1.00 2 31 Rv0703 rplW 50S ribosomal proteinL23 −4.1 1.00 2 32 Rv0704 rplB 50S ribosomal protein L2 −10.1 1.00 2 33Rv0705 rpsS 30S ribosomal protein S19 −9.5 1.00 2 34 Rv0708 rplP 50Sribosomal protein L16 −2.4 0.66 2 35 Rv0709 rpmC 50S ribosomal proteinL29 −4.2 1.00 2 36 Rv0710 rpsQ 30S ribosomal protein S17 −6.2 1.00 2 37Rv0718 rpsH 30S ribosomal protein S8 −3.0 0.99 2 38 Rv0719 rplF 50Sribosomal protein L6 −5.0 1.00 2 39 Rv0721 rpsE 30S ribosomal protein S5−2.4 0.91 2 40 Rv0723 rplO 50S ribosomal protein L15 −2.1 0.58 2 41Rv0988 conserved hypothetical −2.1 0.65 3 protein 42 Rv1182 papA3PKS-associated protein, −5.3 1.00 1 unknown function 43 Rv1183 mmpL10conserved large membrane −2.6 0.96 3 protein 44 Rv1184c conservedhypothetical −3.4 1.00 3 protein 45 Rv1185c fadD21 acyl-CoA synthase−2.1 0.49 1 46 Rv1198 conserved hypothetical −2.0 0.03 3 protein 47Rv1297 rho transcription termination −2.4 0.82 2 factor rho 48 Rv1298rpmE 50S ribosomal protein L31 −2.0 0.33 2 49 Rv1535 hypotheticalprotein −2.1 0.58 16.6 50 Rv1613 trpA tryptophan synthase [alpha] −2.50.92 7 chain 51 Rv1614 lgt prolipoprotein diacylglyceryl −4.6 1.00 3transferase 52 Rv1643 rplT 50S ribosomal protein L20 −2.5 0.93 2 53Rv1794 conserved hypothetical −2.0 0.46 10.5 protein 54 Rv1810 conservedhypothetical −4.4 1.00 10.5 protein 55 Rv1826 gcvH glycine cleavagesystem H −2.5 0.92 7 protein 56 Rv1883c conserved hypothetical −2.4 0.7010.5 protein 57 Rv1886c fbpB antigen 85B, −2.3 0.80 1 mycolyltransferase58 Rv2067c conserved hypothetical −2.1 0.60 10.5 protein 59 Rv2080 lppJlipoprotein −2.7 0.98 3 60 Rv2147c hypothetical protein −2.2 0.22 10.561 Rv2190c putative p60 homologue −3.1 1.00 0 62 Rv2391 nirA probablenitrite −3.5 1.00 7 reductase/sulphite reductase 63 Rv2392 cysH3′-phosphoadenylylsulfate −3.5 1.00 7 (PAPS) reductase 64 Rv2441c rpmA50S ribosomal protein L27 −2.2 0.65 2 65 Rv2840c conserved hypothetical−2.3 0.72 10.5 protein 66 Rv2928 tesA thioesterase −2.0 0.53 1 67Rv2948c fadD22 acyl-CoA synthase −2.8 0.98 1 68 Rv2949c hypotheticalprotein −16.1 1.00 10.5 69 Rv2950c fadD29 acyl-CoA synthase −3.2 1.00 170 Rv2959c some similarity to −2.2 0.67 7 methyltransferases 71 Rv2986chupB DNA-binding protein II −2.4 0.89 2 72 Rv3135 PPE50 PPE-familyprotein −2.0 0.55 6 73 Rv3148 nuoD NADH dehydrogenase −2.4 0.87 7 chainD 74 Rv3152 nuoH NADH dehydrogenase −2.2 0.64 7 chain H 75 Rv3153 nuoINADH dehydrogenase −3.6 1.00 7 chain I 76 Rv3154 nuoJ NADH dehydrogenase−2.5 0.88 7 chain J 77 Rv3377c similar to many cyclases −2.2 0.73 7involved in steroid biosynthesis 78 Rv3456c rplQ 50S ribosomal proteinL17 −3.3 1.00 2 79 Rv3457c rpoA [alpha] subunit of RNA −2.1 0.59 2polymerase 80 Rv3460c rpsM 30S ribosomal protein S13 −2.7 0.96 2 81Rv3461c rpmJ 50S ribosomal protein L36 −2.5 0.78 2 82 Rv3477 PE31PE-family protein −4.8 1.00 6 83 Rv3478 PPE60 PPE-family protein −4.11.00 6 84 Rv3487c lipF probable esterase −4.4 1.00 7 85 Rv3600cconserved hypothetical −2.1 0.63 10.5 protein 86 Rv3680 probable aniontransporter −2.3 0.80 3 87 Rv3686c conserved hypothetical −3.2 1.00 10.5protein 88 Rv3763 lpqH 19 KD lipoprotein antigen −3.2 0.99 3 precursor89 Rv3783 rfbD integral −2.5 0.94 3 membranememebrane protein, ABC-2SUBFAMILY 90 Rv3806c possible integral membrane −2.1 0.62 3 protein 91Rv3822 conserved hypothetical −2.6 0.97 10.5 protein 92 Rv3823c mmpL8conserved large membrane −4.0 1.00 3 protein 93 Rv3824c papA1PKS-associated protein, −3.7 1.00 1 unknown function 94 Rv3825c pks2polyketide synthase −2.2 0.70 1 95 Rv3921c unknown membrane −4.5 1.00 3protein 96 Rv3922c possible hemolysin −4.7 1.00 0 97 Rv3923c rnpAribonuclease P protein −2.4 0.88 2 component 98 Rv3924c rpmH 50Sribosomal protein L34 −3.0 0.99 2 Negative Regulation 99 Rv0079hypothetical protein 3.7 1.00 16.6 100 Rv0129c fbpC2 antigen 85C, 2.70.53 1 mycolytransferase 101 Rv0188 putative methyltransferase 6.4 1.003 102 Rv0211 pckA phosphoenolpyruvate 3.8 1.00 7 carboxykinase 103Rv0233 nrdB ribonucleoside-diphosphate 3.0 0.97 2 reductase B2 104Rv0276 conserved hypothetical 2.2 0.51 10.5 protein 105 Rv0341 conservedhypothetical 5.6 1.00 3 protein 106 Rv0347 conserved hypothetical 4.61.00 3 protein 107 Rv0569 conserved hypothetical 6.4 1.00 10.5 protein108 Rv0570 nrdZ ribonucleotide reductase, 2.7 0.67 2 class II 109Rv0572c hypothetical protein 2.1 0.01 16.6 110 Rv0677c mmpS5 conservedsmall membrane 2.1 0.25 3 protein 111 Rv0805 conserved hypothetical 2.80.92 10.5 protein 112 Rv0823c ntrB transcriptional regulator, 2.6 0.53 9ntrB (NifR3/Smm1 family) 113 Rv0824c desA1 acyl-[ACP] desaturase 2.70.17 1 114 Rv0885 unknown transmembrane 3.0 0.98 10 protein 115 Rv0967conserved hypothetical 2.2 0.43 10.5 protein 116 Rv0968 conservedhypothetical 2.0 0.27 10.5 protein 117 Rv1303 conserved hypothetical 2.30.45 3 protein 118 Rv1304 atpB ATP synthase a chain 2.4 0.64 7 119Rv1332 putative transcriptional 2.0 0.18 9 regulator 120 Rv1461conserved hypothetical 2.1 0.10 10.5 protein 121 Rv1577c phiRV1 possibleprohead 2.4 0.65 5 protease 122 Rv1622c cydB cytochrome d ubiquinol 12.11.00 7 oxidase subunit II 123 Rv1623c appC cytochrome bd-II oxidase 7.51.00 7 subunit I 124 Rv1733c possible membrane protein 2.8 0.77 3 125Rv1737c narK2 nitrite extrusion protein 2.2 0.12 3 126 Rv1738 conservedhypothetical 2.9 0.17 10.5 protein 127 Rv1813c conserved hypothetical9.6 1.00 10.5 protein 128 Rv1846c putative transcriptional 2.4 0.70 9regulator 129 Rv1894c some similarity to 2.2 0.44 10.5 dioxygenases 130Rv1955 hypothetical protein 2.5 0.79 16.6 131 Rv1996 conservedhypothetical 12.7 1.00 10.5 protein 132 Rv1997 ctpF probable cationtransport 3.4 1.00 3 ATPase 133 Rv2031c hspX 14 kD antigen, heat shock2.5 0.04 2 protein Hsp20 family 134 Rv2032 acg conserved hypothetical3.8 0.99 10.5 protein 135 Rv2160c hypothetical regulatory 2.4 0.66 9protein 136 Rv2193 ctaE cytochrome c oxidase 2.3 0.04 7 polypeptide III137 Rv2280 similar to D-lactate 2.2 0.30 7 dehydrogenase 138 Rv2495cpdhC dihydrolipoamide 2.1 0.31 7 acetyltransferase 139 Rv2497c pdhApyruvate dehydrogenase 2.5 0.70 7 E1 component [alpha] subunit 140Rv2557 conserved hypothetical 4.6 1.00 16.5 protein 141 Rv2623 conservedhypothetical 7.1 1.00 10.5 protein 142 Rv2624c conserved hypothetical4.3 1.00 10.5 protein 143 Rv2625c conserved hypothetical 5.8 1.00 3protein 144 Rv2626c conserved hypothetical 5.1 1.00 10.5 protein 145Rv2627c conserved hypothetical 11.5 1.00 10.5 protein 146 Rv2628hypothetical protein 15.0 1.00 16.6 147 Rv2629 hypothetical protein 4.01.00 10.5 148 Rv2630 hypothetical protein 2.0 0.27 16.6 149 Rv2780 aldL-alanine dehydrogenase 8.0 1.00 7 150 Rv2846c efpA putative effluxprotein 2.4 0.47 3 151 Rv3048c nrdG ribonucleoside-diphosphate 2.5 0.772 small subunit 152 Rv3053c nrdH glutaredoxin electron 2.2 0.16 2transport component of NrdEF 153 Rv3127 conserved hypothetical 5.3 1.0010.5 protein 154 Rv3129 conserved hypothetical 7.2 1.00 10.5 protein 155Rv3130c conserved hypothetical 4.4 0.99 10.5 protein 156 Rv3131conserved hypothetical 3.5 0.93 10.5 protein 157 Rv3139 fadE24 acyl-CoAdehydrogenase 3.7 1.00 1 158 Rv3140 fadE23 acyl-CoA dehydrogenase 8.81.00 1 159 Rv3230c similar to various 2.7 0.89 7 oxygenases 160 Rv3675hypothetical protein 2.6 0.76 3 161 Rv3841 bfrB bacterioferritin 6.21.00 7 162 Rv3842c glpQ1 glycerophosphoryl diester 2.3 0.26 7phosphodiesterase 163 Rv3854c ethA probable monooxygenase 2.8 0.91 7

What is claimed is:
 1. An engineered Mycobacterium tuberculosis strainwhose genome comprises a disruption of an rv0348 gene.
 2. An engineeredattenuated Mycobacterium tuberculosis strain whose genome comprises adisruption of an rv0348 gene.
 3. An immunogenic composition comprising apharmaceutically acceptable carrier and an engineered attenuatedMycobacterium tuberculosis strain whose genome comprises a disruption ofan rv0348 gene.
 4. The immunogenic composition of claim 3, furthercomprising a pharmaceutically acceptable adjuvant.
 5. A method ofstimulating an immune response comprising: administering to amammal/subject an immunogenic composition according to claim 3.